Labeling, photonic proteins

In addition to photonic switches, photonic proteins were engineered to contain fluorescent moieties that react to changes in a protein s local environment and to conformational changes. In this section, we review several examples of designing photonic probes that goes beyond simple chemical labeling and cysteine mutagenesis. 

In addition to custom design of photonic proteins, which normally requires site-specific modifications at particular internal residues, TRAMPE can be used to incorporate photonic moieties at nonspecific positions, such as at random lysine residues or only at the N-terminal position of a protein. This approach can be useful in the fields of gene expression, proteomics, and even medical molecular diagnostics. For example, fluorescent labels can be used to monitor in vitro or even in vivo expression of proteins, to build arrays of proteins with which to detect specific protein-protein or drug-protein interactions, and even to detect genetic defects, which are expressed on the protein leveL... 

The FRAP apparatus can also be used in a semi-quantitative manner to measure the surface concentration and subsequent competitive displacement of adsorbed labelled species, such as the fluorescent-labelled protein in the adsorbed layer of a/w or o/w thin films [10]. This can be achieved by focusing the low power 488 nm beam on the film and detection of the emitted fluorescence using the FRAP photon counting photomultiplier. The detected fluorescence signal is proportional to the amount of adsorbed protein at the interfaces of the thin film provided that the incident laser intensity is kept constant. Calculations have proved that the contributions from non-adsorbed protein molecules in the interlamellar region of the film are negligible [12],... 

Ouyang H, DeLouise LA, Miller BL, Fauchet PM (2007) Label-free quantitative detection of protein using macroporous silicon photonic bandgap biosensors. Anal Chem 79 1502-1506... 

More recently, confocal fluorimetry itself has been impressively extended. In particular, the implementation of multi-photon excitation opened the potential to excite different fluorescent labels by a single laser line [47]. This considerably simplified the optical setup of confocal instruments. For example, Heinze et al. [48] described a setup for two-photon excitation confocal fluorimetry where three molecular species were quantified simultaneously using a single laser. When included in screening systems, these spectroscopic advancements enable the quantification of enzymatic reaction rates on several substrates in parallel or, when applied for peptide or protein ligands, the simultaneous measurement of binding affinities on different target receptors. In this way, biopharmaceuticals can be selected on the basis of their specificity and selectivity. As a consequence, undesired side activities can be controlled very early in the hit identification process. 

---