Bioluminescence in analytical chemistry and in vivo imaging☆
Introduction
Bioluminescence (BL) is simply light produced by a chemical reaction in a living organism, usually involving an enzyme/substrate system. In this process, at least part of the reaction products are obtained in an electronically excited state, which can either decay, emitting photons of visible light, or act as sensitizer, passing their energy to another chemical species that will in turn emit light. In other words, there is no difference between BL and chemiluminescence (CL) in terms of conceptual mechanism and luminescence classification.
The BL emission intensity depends on the overall quantum yield of the reaction (ΦBL), which is the product of the chemical yield of the reaction (ΦC), the excited state production yield (ΦEX), and the emission quantum yield of the excited state (ΦF). When compared with conventional CL systems, the peculiarity of a BL reaction is the much higher efficiency of the light emission process. Indeed, the quantum yield of BL reactions can be close to unity, as in the case of the firefly luciferase BL reaction (although this value has been recently questioned), which means that one photon can be obtained (and potentially detected) for each reacting molecule.
Thanks to the high detectability of the luminescence signal, the relatively simple chemistry and the remarkable quantum yields, BL systems have been used in any bioanalytical field where high sensitivity is required, from ATP measurement in hygiene monitoring and food analysis to immunoassays and nucleic acid hybridization assays. The advancements of molecular biology in the last decades have further enlarged the diffusion of BL-based bioanalytical methods. Bioluminescence resonance energy transfer (BRET) assays for monitoring protein-protein interactions, BL whole-cell biosensors for the detection of heavy metals and xenobiotics, and whole-body BL imaging systems for tracking tumor cells and evaluating gene expression in living animals are representative examples of recent BL applications with a largely unexplored potential. Ultrasensitive miniaturized analytical instruments, such as lab-on-a-chip devices, in which BL measurement are performed in ultra-small samples, down to the nanoliter scale, have been also developed by employing integrated light detectors, such as charge-coupled devices (CCDs), CMOS sensors and single photon avalanche photodiodes (SPADs).
Section snippets
Bioluminescent proteins
From a biochemical point of view, all known BL proteins use molecular oxygen to oxidize their substrates to a product molecule in its electronically excited state. In order to obtain an excited-state product, all BL reactions must be strongly exoergonic: for instance, the energy released from the oxidation of D-luciferin catalyzed by firefly luciferase is about 10 times greater than that obtained from the hydrolysis of ATP. In nature, there are more than 30 different chemically unrelated BL
Why analytical bioluminescence?
Luminescence-based techniques, relying on CL or BL systems, offer undoubted advantages over other spectroscopic detection techniques, such as photoluminescence (fluorescence), most noticeably a higher detectability due to the lower non-specific background signal and the absence of interference from excitation light. For the analytical chemist, the most appealing feature of BL is without doubt the high quantum yield, which is about one order of magnitude higher than that of CL reactions. This,
Mutant BL proteins
Molecular biology techniques are extensively used for the improvement of existing BL systems through production of mutated BL proteins with different emission properties.
Instrumentation for BL (and CL) measurements
Both BL and CL analytical applications gained advantage from the recent technological improvements in light detection, especially as concerned the development of ultrasensitive high-resolution CCD cameras for low-light imaging devices and solid-state light detectors for compact device formats.
Bioluminescent proteins as ultrasensitive chemical reagents
Bioluminescent systems can be used, either alone or coupled with other enzymatic reactions, for the detection of a variety of analytes, taking advantage of the high detectability of the BL signal to perform sensitive and fast analyses.
Assays for monitoring protein-protein interactions
Assays where a protein-protein interaction or a protein conformational change is revealed by BL have been developed with exciting perspectives in biosciences.
Immunoassays
Despite the potential advantages of BL over CL, especially for what concerns sandwich-type immunoassays that would greatly benefit from higher label detectability, relatively few enzyme immunoassays with BL detection (BLEIAs) have been reported so far with respect to the high number of immunoassays relying on HRP/luminol CL system. The target analytes of BLEIAs include small molecules (e.g., thyroxine, digoxin, cortisol, thyrotropin), proteins (e.g., prostatic acid phosphatase, prostate
Nucleic-acid hybridization assays
Nucleic-acid hybridization assays are essentially sandwich-type assays, thus they could also take advantage from high label detectability. However, as for BLEIAs, relatively few examples of BL hybridization assays have been reported so far, mainly employing aequorin or obelin as labels and showing detection limits in the low pmol range of target nucleic acid [59]. A new chemical method for the synthesis of firefly luciferase-labeled oligodeoxynucleotides that retained BL activity has been
Cell-based assays
The first cell-based BL assays exploited the spontaneous luminescence of bacterial strains such as V. fischeri and V. harveyi to obtain “living toxicity sensors”. These whole-cell biosensors constitute a well-established and standardized tool for toxicity testing of water and of every sample suspected to be toxic [66]. Evaluation of the toxicity of a sample is very straightforward because it results in the decrease of the BL emission, due to either inhibition of the enzymes involved in the
High-throughput screening
The recent technical advancements in detection devices and automation systems for high-throughput analysis have radically revolutionized the way BL cell-based assays, and in general BL measurements, can be implemented and used [71]. Indeed, luminescence is an ideal detection principle for HTS because it combines high detectability and rapid measurement.
The use of bacterial luciferase has greatly facilitated the development of HTS assays [71]. For instance, a microfluidic HTS assay for sirtuin
Miniaturized devices
Luminescence detectors can be easily integrated into miniaturized analytical devices. The production of micro-opto-electro-mechanical-systems (MOEMSs), i.e., lab-on-a-chip platforms integrating whole-cell biosensors and ultrasensitive photodetectors, is now a reality with unlimited potential applications. As a proof-of-principle, a lab-on-a-chip device was constructed by Elman et al. using genetically engineered bacteria expressing bacterial luciferase as the BL reporter protein [75]. A
In vivo BL imaging
Nowadays, molecular imaging is mostly based on optical, magnetic resonance and nuclear medicine modalities. Among them, optical imaging employing either fluorescent or BL probes is one of the most sensitive technologies and probably represents the most cost-effective and simple procedure, although it cannot be transferred to bedside applications. By means of optical imaging, cellular and molecular events can be monitored in real time and in a quantitative way, with the great advantage of a
Curiosities
Besides well-known uses, BL has been widely used for uncommon applications. For instance, Deforidt et al. investigated the relationship between BL emission, virulence and quorum sensing in pathogenic vibrios [92]. To better elucidate the role of BL in vivo and to investigate human disorders related to circadian rhythm disturbances, human-derived cells showing circadian rhythms of BL have been studied [93]. Dinoflagellate BL in response to hydrodynamic stress was evaluated using a microfluidic
Conclusion
In the past two or three years, new concepts for exploiting BL in the development of bioanalytical tools have been published, strongly suggesting that the fascinating world of BL still deserves continuous promising perspectives. The combination of an easy manageable analytical signal with the possibility of using modern biotechnological tools to purify, clone, and modify BL proteins has led to numerous examples of their use as probes or reporter genes for biosensing technology and for in vivo
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Just as this article was ready for submission, the announcement that the Nobel Prize for Chemistry had been awarded to Osamu Shimomura, Martin Chalfie and Roger Y. Tsien for the discovery and the development of the green fluorescent protein (GFP) made us feel very proud, as were all scientists involved in the study of light emission and related phenomena occurring in nature, from the dark deep of the ocean to the springtime night sky with fireflies flashing. This Nobel Prize is a clear sign of the significance of these phenomena in molecular biology and the development of new analytical tools that are revolutionizing bioanalysis and, more generally, life sciences.