**2.3 ATP-Bioluminescence applications**

100 Bioluminescence – Recent Advances in Oceanic Measurements and Laboratory Applications

Light-producing living organisms are widespread in nature and from diverse origins. The process of light emission from organisms is called bioluminescence and represents a chemical conversion of energy into light. Since the work of William D McElroy showing that ATP is a limiting and key factor of the bioluminescent reaction, research has lead to a better understanding of how light is produced by fireflies (McElroy, 1947; McElroy, 1951; McElroy et al., 1953). The bioluminescence mechanism involving Luciferase enzyme is a multistep process which mainly requires Luciferin substrat, Oxygen (O2), Magnesium cation (Mg++) and ATP (DeLuca & McElroy, 1974; McElroy et al., 1953; Seliger, 1989). ATPbioluminescence using luciferine/luciferase relies on luciferine oxidation by the luciferase and the integrated light intensity is directly proportional to ATP contents. Luciferase converts in presence of ATP and Magnesium firefly D-luciferin into the corresponding enzyme-bound luciferil adenylate. The luciferil adenylate complex is then the substrate of the subsequent oxidative reaction leading to oxyluciferin. The light emission is a consequence of a rapid loss of energy of the oxyluciferine molecule from an excited state to a stable one. This reaction induces the emission of photons with a efficient quantum yield of

Luciferil adenylate complex +PPi

2 Oxyluciferin + AMP+ CO2 + light

Fig. 1. Chemical reactions of the ATP-bioluminescence based on luciferin/luciferase system (PPi:inorganic pyrophosphate, CO2: Carbon Dioxide). Photons of yellow-green light (550 to

Luciferase is a common term used to describe enzymes able to catalyze light emission. Luciferase belongs to the adelynate-forming protein family and is an oxygen-4 oxidoreductase gathering decarboxylation and ATP-hydrolysing main activities. Structural studies have shown that Photinus pyralis Luciferase protein is folded into 2 domains: a large N-terminal body and a small C-terminal domain linked by a flexible peptide creating a wide cleft (Conti et al., 1996). Amino acids critical for bioluminescence phenomenon belong mainly to the N-terminal domain (Branchini et al., 2000; Thompson et al., 1997; Zako et al., 2003). This implies that luciferine-binding site is mediated by conformational change to bring the 2 domains closer. This conformational change is consistent with the study of Nakatsu et al (2006) showing that luciferase from luciola cruciata exists in an "open form" and in a "closed form", the later form creates an hydrophobic pocket around the active site and is responsible of light emission. Two kinds of colored light emission are described for luciferine/luciferase reaction. The typical high energy yellow-green light emission with a peak at 562 nm at pH 7.5 and red light emission with a peak at 620nm when the pH decreases to 5 (Seliger et al., 1964; Seliger & McElroy, 1964). This surprising phenomenon where Luciferase is able to emit light of different colors is not clearly understood but the isolation of colored luciferase variants shows that single amino acid substitution in

**2. ATP-Bioluminescence** 

**2.1 ATP-Bioluminescence principle** 

about 90% (Seliger, 1989; Wilson & Hasting, 1998) (Fig1).

1/ D-luciferin + luciferase + ATP Mg

2/ Luciferil adenylate complex *<sup>O</sup>*

570 nm) are emitted.

**2.2 Luciferase protein** 

With the isolation, cloning and purification of various luciferases from many bioluminescence-producing organisms (bacteria, beetles, marines organisms, etc), bioluminescent assays have been developed and widely used in microbiology to detect bacterial contamination by measuring presence of ATP and in molecular and cellular biology with luciferase as reporter gene to monitor gene expression, protein-protein interaction, etc (Francis et al., 2000; Roda et al, 2004; Thorne et al., 2010). The average intracellular ATP content in various microorganisms has been quantified and ATP has been shown to be a reliable biomarker of the presence of living organisms (Kodata et al., 1996; Thore et al., 1975; Venkateswaran et al., 2003). To be able to specifically detect living organisms by ATP-bioluminescence, the first step is to extract ATP from cells. This step is critical and impacts directly the reliability of the detection (Selan et al., 1992). Chemical solution or physical extraction methods were used in liquid samples (Selan et al., 1992; Siro et al., 1982). Some false negative results were described in few studies (Conn et al., 1975; Kolbeck et al., 1985). Additional studies investigated the cause of false negative results and demonstrated that ATP extraction was not efficient. Indeed, extensive sonication of bacterial samples for instance caused a significant increase of Relative Light Unit (RLU) measured (Selan et al., 1992). Taking into account this limitation, ATP-bioluminescent assay has already proved to provide good detection properties in many areas. Bioluminescent assay is broadly used to monitor air and surface cleanliness and product quality mainly in food industries and in less extent in pharmaceutical industries (Aycicek et al., 2006; Bautisda et al., 1995; Davidson et al., 1999; Dostalek & Branyik, 2005; Girotti et al., 1997; Hawronskyj & Holah, 1999). Studies shows that the level of contamination assessed though surface swabbing, ATP extraction and bioluminescent assay correlate well for 80 % of the samples tested with traditional plate method (Poulis et al., 1993). Availability of sensitive luminometers as well as many commercial ATP-bioluminescent kits has allowed the development of various protocols and applications in industrial microbiology. Currently, ATP- bioluminescence is an accepted and common technology used to monitor contamination in areas such as food and beverage, ecology, cosmetic, and clinical (Andreotti & Berthold, 1999; Chen & Godwin, 2006; Davidson et al., 1999; Deininger & Lee, 2001; Frundzhyan & Ugarova, 2007; Miller et al., 1992; Nielsen & Van Dellen, 1989; Selan et al., 1992; Yan et al., 2011).
