Analytical and biotechnological use of bioluminescent proteins

Recently there has been a significant increase in the field of bioluminescent (BL) reporter genes, providing inroads to in vitro and in vivo monitoring of several biological processes (e.g. gene expression, protein-protein interaction and disease progression) and, in general, bioanalytical tools in medical, pharmaceutical and environmental fields. Firefly luciferase is by far the most commonly used BL reporter, although alternative BL proteins cloned from other organisms or obtained by random and site-directed mutagenesis are now being explored to expand the applicability and analytical performance of BL assays. The availability of BL proteins with red-shifted emission and high thermostability, i.e., P. pyralis and L. italica luciferase mutants, paved the way to multiple labelling systems, advanced whole-cell biosensors, multiplexed assays and high-content screening procedures based on BL. The high detectability of the BL signal makes it suitable also for miniaturized bioanalytical devices (e.g., high-density-well microtiter plates, microarrays and microfluidic) for the high-throughput screening of genes and proteins in small sample volumes. Patterns of immobilized biosensors generated by photolithography and microfluidic technologies could be used to fabricate micro cell chips for high throughput screening and point-of-care testing (POCT). Elegant approaches, relying on Bioluminescence Resonance Energy Transfer (BRET) systems and split reporter protein complementation reconstitution strategies, have been explored to investigate protein-protein interactions and study biological pathways. In addition, BL proteins have been also conjugated to quantum dots to obtain a new class of BL probes for multiplexing in vivo molecular imaging. Thanks to its high sensitivity and to the availability of new red- and near infrared-emitting BL proteins, in vivo BL molecular imaging is emerging as one of the leading imaging technologies in the areas of cancer biology, cell biology, gene therapy and stem cell research. The expression of a BL protein can be put under the control of tissue-specific regulatory elements allowing non-invasive imaging of physiological and pathological processes like differentiation, apoptosis, tumour progression, and inflammation, even in a 3D fashion by means of BL tomography, which allows 3D BL source reconstruction. All these BL features, associated to instrumental and technical advancements in miniaturization, sensitivity and resolution of light detection devices, will certainly further expand the application of BL proteins towards the development of POCTs, high throughput screening procedures and ultra-sensitive BL molecular imaging for in vivo monitoring of physio-pathological processes.