Chemiluminescence in Analytical Chemistry

agents also allows differentiating somatic and bacterial cells, disrupted by mild and strong agents, respectively. In any case the effects of detergents on luminescence must be taken in account.

Beside that of Photinus pyralis, other luciferases have been described in the literature such as those from Luciola cruciata, Luciola lateralis [122], and Luciola mingrelica. Among them luciferase from the latter one has been employed for analytical purposes [123].

2.1.1Biomedical Applications

In the study of biological alterations or during the clinical treatment of several pathologies it can be useful to evaluate the cells’ content of ATP, which is often directly related to the alterations under study. ATP determination can be used, for example, in the diagnosis of transformed cells: in general, cancer cells have low ATP levels and their ATP values fall with the length of culturing, but in polyploidal cells the ATP level increases with the polyploidy. Measurements in single cells have proved to be particularly informative in resolving the sequence of events occurring in cells exposed to pathological insults. A great cell-to-cell variability was found in the time course of the ATP decline in response to metabolic poisons. For example, ATP changes were monitored in single hepatocytes, at a concentration of 1 mM or lower, allowing the monitoring of chemical hypoxia [105].

Analogously ATP BL can be used for cytotoxicity assays, i.e., to determine cell response to antibiotics [91], to antineoplastic agents, even in association with ionizing radiation [100], and to the solvents commonly used for solvation of xenobiotic agents [106]. The assay, compared to the classical Ames test [104], showed a very good correlation between the BL assay and plates method. The strong potential of ATP BL to become a clinical assay for chemosensitivity testing therefore appeared clear. It proved to be a highly quantitative assay, which measures cellular response of the cultured cells evaluating cellular ATP instead of counting colonies. The assay was found to be reproducible and reliable and to have a clinical applicability rate of more than 90%, making it well suited for clinical application [98, 99].

ATP is an ideal indicator of cell viability. Blood or blood cell concentrates prepared for transfusion are stored for periods of a few days to several weeks in the blood bank. Viability checking of the blood cells is necessary to avoid posttransfusional reactions [94]. This quality control of the conserved red blood cells and platelets can easily be performed by measuring the ATP concentration as an expression of their integrity. By the same measurement it was possible to confirm the diagnosis and monitor the treatment effects in various cases of platelet disease [97]. The possibility of determining cells’ viability can be exploited to examine more free cells or tissue, as in the spermatozoa viability test, based on the correlation between ATP content and mobility.

Blood platelets release ATP, ADP, serotonin, and other compounds and this plays an important role in thrombosis and hemostasy. To study the mechanism of the release, in vitro release of ATP was followed using the firefly luciferase luminescence method [124].

Firefly luciferase, together with luciferases from other organisms, can be used as the labeling enzyme in immunoassays and nucleic acid assays [25]. Recently, a highly sensitive BL ELISA using firefly luciferase was applied to thyreo-

tropine detection [125] and a BL homogeneous enzyme-binding assay for biotin detection was developed [59]. These are among the few applications reported on this topic. Actually, the enzyme proved to have some problems when employed in this application, being not very stable, and expensive; large batch-to-batch variations were also recorded. To develop such immunoassays, several strategies must therefore be applied, making them not competitive compared to other labeling systems [21, 22]. In the past some luciferins, when derivatized with 6-N- acetyl-L-phenylalanine or o-phosphate, were used in immunoassays [126–128]. In the latter case the reaction with alkaline phosphatase, acting as the labeling enzyme, allowed release of the free luciferin that reacts with subsequent light emission.

Luminometric assay of ATP can also be applied to measurement of other substrates such as ADP, AMP, and ATP-specific enzymes. Other examples are reported in Table 4.

2.1.2Rapid Microbiology

The obvious presence of ATP inside bacterial cells allows setting up new microbiological assays, quicker, easier, more sensitive but at the same time offering the same reliability as classical microbiological plate count. The main difference between the classical plate count method and ATP BL is that BL is able to detect all the cells (bacteria, fungi, yeast, somatic, etc.), both alive and stressed, i.e., not capable of reproducing in that medium, while the plate method measures only the cells that are alive and able to reproduce. For this reason ATP can be used as indicator of biological contamination in different fields [68]. The amount of ATP per cell is essentially proportional to the intracellular volume; consequently most bacterial cells contain around 2 10 18 mol ATP/cell (approx. 10 15 g/cell). However, differences in ATP content were found in different bacterial species and in yeast, for which the amount of ATP is 100–1000 times higher than for bacteria. When a cell dies from natural causes, intracellular enzymes rapidly degrade ATP. If cells are killed in a way that also inactives the enzymes, some ATP may appear even outside cells [129, 130].

The sensitivity obtained with ATP BL is around 1000–10,000 colony-form- ing units (CFU) per milliliter. Better detection was obtained by determining adenylate kinase instead of ATP:10–1000 cells/100 L [71].

Since the U.S. Department of Agriculture, Food Safety and Inspection Service (USDA-FSIS) published the pathogen reduction hazard analysis and critical control point (HACCP), this system has been frequently applied in the quality management and assurance field to increase safety of foods. This is achieved by monitoring and controlling all the steps of foods processing, and especially checking the surfaces in contact with food. Generally, microbiological methods to ver-

ify that the processing plant, equipment, and products have been properly sanitized are time consuming and expensive. There has consequently been considerable interest in rapid microbiological enumeration methods not only in agriculture and food industries but in a wide list of fields: environmental quality control, monitoring systems at sludge and sewage water plants, pharmaceutical and cosmetics industries, vitality and quality of biomasses involved in fermentation processes, bacterial contamination in spaces otherwise sterile (surgical rooms), microorganisms’ contribution to alterations affecting artistic works. ATP BL is one of these alternative methods and has already been widely applied (Table 4). Several commercial kits are available and also directly usable in the field with portable instruments. When ATP is found on a surface, this indicates it is not perfectly clean; the contamination may come from microorganisms, food residues, or human contact. The latter two of these sources can both provide an ideal environment for bacterial growth and neither of them can be detected by conventional microbiological methods. The advantage of ATP measurement in the evaluation of cleanliness is its ability to detect these product residues and organic debris in addition to living microorganisms. At the same time, the fact that the method does not differentiate between ATP from different sources can be a limitation, unless additional selective steps are performed. When ATP BL is employed in monitoring sanitation, it is important to consider that cleaning agents and sanitizers can interfere with ATP detection [81].

Monitoring of ATP levels also proved useful to quantify air contamination in the work environment [111]. Comparison with other analytical methods shows the usefulness of the BL assay because of its rapidity, in spite of being aspecific [131].

ATP measured by luciferin-luciferase BL assay was used to examine the effect of toxic substances on whole microbial communities in activated sludge mixed liquid samples [114]. It was used to detect whether wastewater had an effect on the biodegradation capability of the resident population of microorganisms. Actually ATP BL represents an important rapid toxicity test that utilizes waste treatment natural microorganisms to determine the toxicity of wastes discharged to the sewer [132].

Another interesting application of the ATP assay is related to alterations affecting artistic stonework, which can be due to various factors. Air pollution is recognized as the major cause, but microbiological attack also plays an important role. These alterations have been observed on several Italian monuments such as Scalone dei Giganti in Venice, the facade of Certosa di Pavia, the Duomo di Siena, and the Duomo di Orvieto, always on Carrara marble. For more than 50 years studies have been carried out on the causes of these alterations and only recently has their biological origin been taken into consideration. Bioluminescent assays were used to confirm the presence of microorganisms [113].

2.1.3Firefly Luciferase as Reporter Gene

Genetically modified microorganisms hold great promise for many applications including analytical uses in the environmental, industrial, and agricultural fields [24, 25, 41]. A key component of this technique is a reliable detection method, specific and extremely sensitive, to study the fate of the modified organism and/ or engineered DNA in environmental samples or field tests. The firefly luciferase gene has been proposed as an ideal marker for genetically modified organisms to be released into the environment, having the requested characteristics. In addition, the bioluminescent assays are extremely fast and easy, relatively inexpensive, there is no phenotype in the absence of luciferin, and luc-tagged cells can be detected by a variety of complementary methods: this provides flexibility in choosing the appropriate method for specific applications [133]. The methods for detection of the luc marker gene can rely on cultivation of the tagged organisms and detection of whole cells, detection of cell extracts, or detection of the gene by polymerase chain reaction (PCR), each one having advantages and disadvantages [133]. In particular, the luciferase gene marker is ideally detected by PCR amplification since it is not naturally present in the microbial population.

An entirely different class of luciferase enzyme has been isolated from the firefly Photinus pyralis and related species, comparing the properties of the recombinant ones to the crystalline native enzyme [134]. The firefly luciferase gene luc was shown to act as a specific marker for monitoring of genetically modified bacteria, yeast, and plant leaves [135, 136]. The eukaryotic enzyme was approximately 10 times more sensitive than the corresponding bacterial one. Specific strains of very common bacteria, for example Escherichia coli, were often used as the recipient strain for recombinant plasmids. Using a multicopynumber plasmid as vector for luc gene often enhances sensitivity in the detection of luc-tagged cells.

An interesting example of sensitive and convenient biosensing of environmental pollutants was developed by fusing the firefly luciferase gene to the TOL plasmid of Pseudomonas putida. This plasmid encodes a series of enzymes for degradation of benzene and its derivatives. The gene fusion resulting plasmid was used to transform E. coli, and then applied to the environmental biosensing of benzene derivatives: the expression of luciferase was induced in the presence of aromatic compounds and at low detection limits (5 M for m-xylene) [137]. In a second step the transformed E. coli, bearing firefly luciferase gene fused to TOL plasmid, was immobilized at one end of a fiberoptic demonstrating the possibility to obtain a luminescent remote biomonitoring device for protection against environmental deterioration [138].

Since the genetic code is universal among all living organisms, the coding region of a bacterial enzyme can be successfully expressed as a reporter gene not only in microorganisms, but also in animal cells and plants. The luc gene

262 Girotti et al.

Luciferase

FMNH2 RCHO O2 → FMN RCOOH H2 O Light (2)

Bacterial luciferase is a heterodimeric enzyme of 77 kDa that can be included, in general, under the group of the pyridine-nucleotide-linked systems. Bacterial luciferase is highly specific for FMNH2, but the enzyme also shows weak activity toward other flavins. Only aliphatic aldehydes with a chain length of eight or more carbon atoms are effective in the luminescent reaction. The total light production is proportional to the amount of each of the substrates (O2, FMNH2, RCHO) when they are present in limited quantities. Generally, the emission spectra are characterized by a broad emission with a maximum near 478– 505 nm [14]. Various substances of biological interest and enzyme activities can be analyzed by coupling the luciferase and the oxidoreductase to a third reaction, which produces or consumes NADH or NADPH.

2.2.1Applications of the Isolated Bacterial Luminescent System

The possibility of isolating the components of the two above-reported coupled reactions offered a new analytical way to determine NADH, FMN, aldehydes, or oxygen. Methods based on NAD(P)H determination have been available for some time and NAD(H)-, NADP(H)-, NAD(P)-dependent enzymes and their substrates were measured by using bioluminescent assays. The high redox potential of the couple NAD /NADH tended to limit the applications of dehydrogenases in coupled assay, as equilibrium does not favor NADH formation. Moreover, the various reagents are not all perfectly stable in all conditions. Examples of the enzymes and substrates determined by using the bacterial luciferase and the NAD(P)H:FMN oxidoreductase, also coupled to other enzymes, are listed in Table 5.

2.2.2Applications of Whole Luminescent Bacteria

Toxic pollutants are found everywhere, in water, in the air, and in the soil, causing environmental damage. Therefore, it is increasingly important that their early detection but at the same time the use of animals for toxicity testing has come under critical surveillance. For ethical and economic reasons various techniques have been developed and proposed as potential alternatives, among them the luminescent bacteria toxicity test [167]. Luminescent bacteria emit light when they find themselves in an optimal environment. In vivo luminescence is a sensitive indicator of xenobiotic toxicity to microorganisms because it is directly coupled to respiration via the electron transport chain, and thus reflects the metabolic status of the cell. If noxious substances are present, the luminescence decreases. The higher the degree of toxicity, the less the amount of light emitted by the bacteria. Thus, the presence of toxic substances can be evaluated and several commercial kits and dedicated instruments are now available. The luminescent

bacteria toxicity test, compared with other bioassays, proved that its average sensitivity is well within the same order of magnitude as the other tests in evaluating organic or inorganic pollutants [168–170]. It is, however, acknowledged that the ‘‘battery of test’’ approach, utilizing several different short-term biological tests, would be preferred in any monitoring scheme.

Luminescent bacteria also allow detection of the carcinogenic effect of genotoxics. A dark mutant of a Photobacterium or Vibrio strain that can revert back to luminescence at an increased rate in the presence of base-substitutes or frameshifts agents, DNA-damaging agents, DNA synthesis inhibitors, and DNA intercalating agents can be employed [171, 172].

Additional examples of the analytical applications of BL bacterial bioassays are listed in Table 6.

2.2.3Bacterial lux Genes as Reporter Gene

Cloning and expression of the DNA coding for luciferases from different luminescent organisms have provided the basis for the rapid expansion in the knowledge of molecular biology of luminescence [195, 196] and in its use. It was found