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that significant differences exist between the lux systems from luminescent strains of the same species. In general, the bacterial lux genes can be routinely transferred into E. coli by transformation using a variety of different plasmids. For high expression a promoter is usually required on the plasmid [195, 197].

The ability to introduce the lux phenotype into different bacterial species provides a convenient method for rapidly screening in a simple and sensitive way the presence of specific bacteria and for monitoring their growth and distribution in the environment [198]. Another application of transformed bacteria deals with specific susceptibility in toxicity tests: the presence of agents that disrupt or kill the bacteria destroys the metabolism, thus eliminating light emission. Some examples are listed in Table 7.

3. IMMOBILIZED BIOLUMINESCENT SYSTEMS

The extreme sensitivity and specificity of the light-producing systems may be improved by using immobilized enzymes on solid supports. The immobilized enzymes are often more stable than the soluble forms and they can be used for several analyses, reducing costs. The sensitivity of the assays is generally increased, owing to the creation of a microenvironment with locally high concentrations of the involved reagents. Min et al. [221] reported eight times higher BL activity of immobilized FMN:NAD(P)H oxidoreductase and luciferase than the free enzymes. They developed inexpensive and sensitive biosensors for measuring the substrates of NAD(P)H-dependent enzymes, immobilizing the two in vivo biotinylated enzymes on avidin-conjugated agarose beads.

Various supports are used for immobilizing proteins, among them polyacrylic hydrazide, controlled-porosity glass beads, amino alkylated glass rods, cellophane films, cellulose films, collagen, sepharose, epoxy methacrylate, acyl- azide-activated-collagen strips, and nylon [222]. Several procedures are available for coupling proteins with those supports. In general, the chemical methods give better yields of active immobilized enzymes than the physical ones, whose main disadvantage is the fragility of the binding, sensitive to temperature, pH, and ionic strength variations. The chemical methods use various substances, such as glutaraldehyde, to covalently bind the protein through amino acidic residues. The most important factor in the choice of the suitable method of immobilization is conservation of the enzymatic activity. New methods of immobilization minimizing enzyme inactivation are continuously being investigated. Recently, a new protocol for luciferase immobilization was reported, which does not require prior silanization and glutaraldehyde activation, thus saving preparation time [223]. The method is based on coimmobilization by adsorption of luciferase and poly-L-

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lysine on nonporous glass strips. Luciferase immobilized in this way has minimal variation in intersample activity and good stability.

Another important aspect of immobilized enzymes is that they can be incorporated into flow cells where they can be used for multiple assays. The continu- ous-flow format offers greater possibilities than a single-batch system because it leads to rapid and sensitive assays. Flow assays are characterized by extremely accelerated kinetics: a very high surface-area-to-volume ratio is obtained and the reactions do not have to rely on passive diffusion to bring reagents together. Many analytes can be detected at pmol levels, with good precision and a wide range of linearity. Moreover, the analysis time is reduced to a few seconds and small specimens are needed.

Biosensors are generally described as probe-type devices made up of a selective biological layer with very sharp molecular-recognition capacity and of a physicochemical transducer [224]. Enzymes, antibodies, and nucleic acid sequences are reported as the most common biological element used in biosensors, because of their strong affinity for distinctive target molecules. Whole bacteria were also used to develop microbial biosensors [225]. As a transducer, strong interest was shown in optical systems and, between them, in those based on luminescence: they do not require light sources and monochromators, and they display selectivity, sensitivity, and versatility. Coulet and Blum developed several biolu- minescent-based sensors using commercially available preactivated polyamide membranes to immobilize luminescent enzymes. The tubes connected the enzymatic membrane to an 8-mm-diameter, 1-m-long glass fiber bundle through a screw cap [19, 226, 227]. The light generated by the enzymatic reaction in the presence of the target analytes was conducted to the photomultiplier tube (PMT) of a luminometer. This fiberoptic sensor proved to be suitable for any kind of luminescent enzymes, allowing detection of several analytes.

Table 8 lists analytes that were determined by using immobilized luminescent enzymes [20].

A typical example of these analytical systems is a manifold using bacterial luciferase for L-phenylalanine assay [228] developed with two separate nylon coils, as shown in Figure 3. The first one contained the specific L-phenylalanine dehydrogenase (L-PheDH) enzyme.

L-PheDH

L-Phenylalanine NAD ← → L-phenylpyruvate

NADH H NH4

The second one contained the bacterial bioluminescent enzymes. This system made it possible to reach a detection limit of 0.5 µmol/L.

Sensitive flow-injection analyses of aspartate, glutamate, 2-oxoglutarate, and oxaloacetate were developed using immobilized bacterial luciferase enzymes.

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Table 8 Analytical Performance of the Immobilized Bioluminescent Enzymes
		Detection	Imprecision
	Range	limit	Content
Analyte	(pmol)	(µmol/l)	(pmol)	RSD (%)
NADH (B)a	1–2500	0.1	2.5	3.7
NADPH (B)	5–1500	1	10	5.3
α-Bile acid (B)	1–1000	0.5	2.7	5.9
3α-Bile acid (B)	1–1000	0.5	2.8	7.1
12α-Bile acid (B)	10–10000	1	5.2	6.3
Ethanol (B)	50–2500	1	100	8.3
Glycerol (B)	50–500	2	70	7.2
Acetaldehyde (B)	50–1000	2	100	6.8
L-Alanine (B)	5–500	0.5	75	7.4
Branched-chain	20–2000	0.5	75	7.8
amino acids (B)
ATP (L)	0.05–100	0.02	1.5	8.0
ADP (L)	0.6–100	0.1	15	8.2
ATP (M, r-LM)	1–500	0.3	10	9.3
ATP (r-LN)	0.3–100	0.06	10	5.2
L-Lactic acid (B)	1–500	0.1	7	5.2
D-Lactic acid (B)	10–500	5	50	7.8
L-Glutamic acid (B)	50–1000	10	500	3.7
L-Phenylalanine (B)	1–100	0.5	50	4.3
Magnesium (L)	0.05–6.7	50	0.67	4.5
Magnesium (r-LN, M)	0.01–6.7	10	0.99	2
Lactate dehydrogenase (B)	3–2000a	1a	151a	8.8
Creatine kinase (L)	0.1–100a	0.1a	17a	8.2

a B, bioluminescent bacterial system on nylon; L, bioluminescent firefly system on nylon; M, bioluminescent firefly system on methacrylate beads; r-LM, recombinant firefly luciferase on methacrylate beads; r-LN, recombinant firefly luciferase on nylon.

a mol/min L.

Precision was generally excellent, and sensitivities were 100–1000-fold higher than with spectrophotometric methods. The immobilized-enzymes preparations were stable for several months and each reactor could be used for 600–800 analyses [229].

A flow assay was reported for determination of inorganic pyrophosphate: a pyrophosphatase was coimmobilized with luciferase on Sepharose beads with continuous flow of saturating concentrations of substrates. The instrument allowed automation with a throughput of approximately one sample every 4 min.

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Figure 3 Example of a typical luminescent flow sensing device [manifold for pesticide chemiluminescent flow assay with one (A) or two (B) columns using immobilized enzymes]. A immobilized dehydrogenase (Phe-DH) 1.0 m coil; B immobilized bioluminescent enzymes 0.5 m coil.

Less than 1 pmol pyrophosphate was determined in a volume of 20 L with a coefficient of variation approximately equal to 4% [230].

A continuous-flow system was developed for the assay of magnesium (II) in serum, drugs, and beverages, using firefly luciferase (luc) or recombinant luciferase (r-luc) from E. coli, immobilized both enzymes on nylon coil and on epoxy methacrylate [231]. The volume of the analyzed specimens was 10 L with a sample assay rate of 20 samples per hour and no carryover in the system. The detection limit of the assay was 0.05 mmol/L for LUC-nylon coil and 0.01 mmol/ L for LUC-Eupergit column and r-luc nylon coil. No interference from ions present in the samples (Ca2 , Mn2 , Fe2 , Cu2 , Zn2 , Co2 ) was detected. The nylonimmobilized enzymes have a relatively high stability (1–4 months), despite low recovery in terms of activity with respect to the soluble forms; Eupergit C gave better sensitivity and activity recovery, but lower enzyme stability (3 days–1 month).

Other analytes recently determined by means of immobilized bioluminescent enzymes are europium(III) [232], D- and L-lactate in beer [233], and D- sorbitol [234]. A novel approach of the sensing layer design was reported for

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this latter assay. In biosensors based on multienzymatic sequential reactions, the different enzymes are usually coimmobilized on the same support. Compartmentalization of the sensing layer is proposed as an alternative, enabling significant improvement in the response of the biosensor. The enhancement was assumed to result from hyperconcentration of the intermediate product, common to the two sequential reactions. Such an approach proved applicable to other systems [235].

Flow sensors were also used coupled with microdialysis probe [236], providing several advantages: the reaction took place in few minutes allowing continuous analysis; the sensitivity was in the order of pmoles; the microdialysis probe allowed biological specimens to be drawn without proteins or macromolecules. This technique can be extended to the analysis of analytes that need to be detected continuously such as during therapy monitoring, or in emergency care units.

The variety of assays reported here displays the great versatility of luminescent detection systems. As already described, in all luminescent systems the main advantages are the high sensitivity and specificity, which reduce to the minimum the sample treatment, and the ease of use of the reagents and the luminometer. Immobilized systems greatly reduce the cost per assay; on the other hand, their preparation requires expertise, especially in the surface activation step on nylon tubes.

4. OTHER BIOLUMINESCENT SYSTEMS

As previously mentioned, luminescent reactions occur in almost all zoological kingdoms. The literature is therefore full of papers reporting bioluminescent systems other than those just described [49, 237].

Cloning of the cDNAs that code for several luciferases from a bioluminescent click beetle, Pyrophorus plagiophtalatus, dates back to 1989 [238]. The clones code for luciferases of four types, distinguished by the colors of BL they catalyze. Owing to these different colors, these clones may be useful when multiple reporter genes are needed. An application was reported in which the different wavelengths allow both a target and control signal to be incorporated into each cell, providing a means of differentiating between specific effects of a genetic sensing system and other nonspecific interfering influences [239].

The oral bacterium Streptococcus mutans, containing the luciferase gene of Pyrophorus plagiophtalatus, was used as rapid assay to estimate the effects of various antimicrobial treatments [93].

Pholasin is the protein-bound luciferin from the bivalve mollusc Pholas dactylus. This substrate reacts with its luciferase and molecular oxygen to produce light. The photoprotein, commercially available, undergoes an oxidative

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reaction to oxypholasin in the presence of superoxide anion, peroxidases, or other oxidants. Actually, it acts as an ultrasensitive detector of reactive oxygen species, such as those derived from activated leukocytes. Therefore, pholasin is a useful tool to quantify inflammation and infections, in particular through recognition of granulocyte conditions: quiescent, activated, prone to degranulation, degranulated, and upor down-regulated in response to receptor stimulants [240].

Cypridina luciferin analogs are widely used for several analytical applications (determination of substrates, enzymes, active oxygen species such as superoxide), but they are mainly related to CL [241, 242].

Other bioluminescent microorganisms such as dinoflagellates have some applications, but generally only for teaching purposes [243].

Some additional luminescent systems of some importance are further described in detail.

4.1 Aequorea victoria

Aequorea victoria is a hydromedusan jellyfish found mainly at Friday Harbor, Washington [244], which emits a greenish light (λmax 508 nm) from the margins of its umbrella. The bioluminescent system consists of two proteins, a calciumbinding photoprotein named aequorin and a green fluorescent protein (GFP). As a consequence of the binding of calcium ions, the aequorin reacts, in vitro, yielding blue light, a blue fluorescent protein (BFP), and carbon dioxide. In the jellyfish, where aequorin is considered to be closely associated with GFP, the excited state of BFP undergoes radiationless energy transfer to GFP, which than emits green light relaxing to its ground state (Fig. 4.)

4.1.1Aequorin

Aequorin is the most widely studied of the Ca2 - binding photoproteins described to date. It is composed of an apoprotein (apo-aequorin; 189-amino-acid residues), molecular oxygen, and a luciferin named coelenterazine. The protein contains three EF-hand Ca2 -binding sites, and when Ca2 occupies these sites, aequorin is converted into an oxygenase (luciferase), which oxidizes coelenterazine. The latter is converted to a highly unstable dioxetanone intermediate, which releases carbon dioxide to form the excited phenolate anion of coelenteramide. Coelenteramide remains noncovalently bound to the protein, emitting blue light when relaxing to the ground state [244].

As light emission from aequorin is dependent on Ca2 , the protein has been widely employed for determination of this ion. In particular, the protein was used in the past in the highly sensitive measurement of intracellular calcium concentration in several kind of cells [16]. More recently immobilized aequorin was used to develop an optical biosensor for measurement of calcium ions in complex

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Figure 4 Reaction scheme for light emission of aequorin.

matrices such as human serum and milk samples. A porous sol-gel glass environment was used for the immobilization, having negligible effects on the protein stability and calcium-ion-binding activity [245].

Cloning of the gene encoding apo-aequorin synthesis allowed recombinant expression of the photoprotein [246], which led to genetic transformation of bacteria, yeast, plant, and animal cells. Several applications have arisen with the use of recombinant aequorin. One of the most interesting was the development of methods to assay Ca2 in a discrete subcellular domain [247]. A further application was the use of recombinant aequorin as a quantitative label in different analytical assays. Several characteristics make aequorin an ideal label: it can be readily conjugated, it generates a high signal-to-noise ratio with a broad range of sensitivity, and the signal is generated rapidly [244]. Lizano et al. pointed out the importance of aequorin as a label in competitive binding assays for biotin [248]. The interaction of the vitamin biotin with the binding protein avidin, which is characterized by strong affinity, is currently employed to enhance the sensitiv-

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ity of competitive binding assay in a variety of samples. It was demonstrated that the Ca2 -triggered luminescent reaction of biotinylated aequorin can be inhibited by the presence of avidin; the reduction of bioluminescent intensity is correlated to the biotin concentration in a sample [249]. The reported assay had a detection limit of 10 14 M with high reproducibility. The same technique was applied to the detection of prostate-specific antigen (PSA) mRNA [250]. PSA mRNA from a single cell, in the presence of one million cells that do not express PSA, was detected with a signal-to-background ratio of 2.5 and typical CVs of 6%. Recently this aequorin BL was also used for detecting biomolecules in picoliter vials [251], which were fabricated on glass substrates using a laser ablation technique. The assay in such small-volume vials can find potential applications in a variety of fields, such as microanalysis and single-cell analysis, where the amount of the sample is limited.

The aequorin label, commercially available, proved to be useful in clinical immunoassay [252] and for the detection of nucleic acids. Aequorin was used to develop a rapid diagnostic assay for qualitative evaluation of cytomegalovirus presence in clinical samples [253]. The assay could be performed in less than 2 h and is amenable to automation and to processing hundreds of samples per hour. In a recent study aequorin-based bioluminescent technology was employed for direct quantification of cytokine mRNA by reverse transcriptase PCR techniques (RT-PCR) and for the investigation of induction of human cytokine expression [254]. The bioluminescent assay proved to be 30to 60-fold more sensitive than radioimaging in detecting human IL-2 and CD3-delta amplicons and allowed processing of hundreds of RT-PCR samples per day.

4.1.2Green Fluorescent Protein

Green fluorescent protein (GFP) is made up of 238 amino acid residues in a single polypeptide chain. The fluorophore is formed by the cyclization and oxidation of three amino acids-Ser-Tyr-Gly [255]. Wild-type GFP absorbs blue light (peaks at 395 nm and 475 nm) and emits green light with a maximum at 509 nm. Several characteristics, together with the molecular cloning of GFP cDNA [256], have led to widespread use of GFP in a variety of fields, including environmental monitoring, cell biology, and molecular biology. Despite being rather thermosensitive, GFP is very resistant to denaturation and does not require any cofactor or substrate. Mutational analysis of GFP has generated a variety of GFP, which differ from the native protein, such as increase in brightness and in stability. As a consequence, use of GFP has even increased.

One of the main applications of GFP is as reporter gene. GFP has been expressed in a variety of organisms, including animals [257], plants [258], bacteria [259], and viruses [260], for monitoring gene expression. The attractiveness unique to GFP as a reporter allows nondestructive in vivo fluorescence visualiza-

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tion. GFP can also be employed to investigate organelle structure and can function as a protein tag, as it tolerates N- and C-terminal fusion to a variety of proteins, many of which retain their native functions.

GFP has also been proposed as a successor to the Ames and SOS chromotest. Billinton et al. [8] obtained a reporter system, employed as genotoxicity biosensor, that uses eukaryotic cells (the baker yeast Saccharomyces cerevisiae) instead of bacteria. The strain produces green fluorescent protein, codon optimized for yeast, when DNA damage has occurred. It was demonstrated that the reporter does not falsely respond to chemicals that delay mitosis, and responds appropriately to the genetic regulation of DNA repair.

In a recent work [261] a recombinant herpesvirus, PrV, expressing a genetically modified version of the cDNA encoding gfp was constructed. GFP proved to be useful for monitoring the virus infection in cell culture and in rat brain tissue and can be used to follow the spreading route of the virus in the nervous system, which corresponds to the synaptic linkage of the neurons. The generated viral strain, with the virulence appropriately reduced, is also potentially applicable as a vehicle for expressing GFP in permissive cells, including use for transneural tract tracing.

Regarding environmental applications, GFP appears to be ideally suited for in situ detection of specific bacteria in environmental samples. GFP as a marker has the advantage of being stable during starvation conditions, which is a common state for bacteria in natural environments. Moreover, the fluorescence intensity of GFP is so strong that even bacteria with a single copy of gfp can be detected [262]. A gfp-transformed Mycobacterium smegmatis strain was employed to investigate the impact of biofilms on pathogen persistence in potable water [263]. Biofilms are ubiquitous in drinking-water-distribution plants. The attachment and retention of the gfp-transformed strain was monitored in laminar flow cells exposed to different concentrations of chlorination. Using a transformed organism allowed tracking its response to disturbances in its environment in real time.

4.2 Obelin

Obelin is a Ca2 -activated bioluminescent photoprotein that has been isolated from the marine polyp Obelia longissima. Binding of calcium ions determines a luminescent emission. The protein consists of 195 amino acid residues [264] and is composed of apoobelin, coelenterazine, and oxygen. As aequorin, it contains three EF-hand Ca2 -binding sites and the luminescent reaction may be the result of coelenterazine oxidation by way of an intramolecular reaction that produces coelenteramide, CO2, and blue light. As for aequorin, the luminescent reaction of obelin is sensitive to calcium and the protein was used in the past as an intracellular Ca2 indicator. More recently, the cloning of cDNA for apoobelin led to the use of recombinant obelin as a label in different analytical systems.

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Recombinant obelin was derivatized with biotin and employed in avidinbiotin immunoassays for detection of biotinylated targets immobilized on microtiter wells. Frank and Vysotski [265] applied this technique to detect alpha-feto- protein (AFP), a level of monitoring widely used for early cancer detection and prenatal diagnosis of some genetic diseases. The results obtained were found to be similar to those obtained for radioimmunoassay. Another method, based again on obelin BL, was successively reported for the assay of alpha-fetoproteins and human luteinizing hormone [266]. A bifunctional protein (proZZ-Obe) was constructed, which has both the luminescent activity of obelin and the ability to bind mammalian IgG. It was demonstrated that the chimeric protein proZZ-Obe can be used as a universal marker to determine almost any antigen in a sandwichformat immunoassay with the F(ab)2 fragment of immunoglobulins.

Recently it was found that obelin mRNA can be a useful tool for evaluating the efficiency of cell-free translation and for screening of translation inhibitors.

4.3 Renilla Luciferase

Renilla luciferase is a monomeric protein contained in the bioluminescent sea pansy Renilla reniformis. The luciferase catalyzes the oxidation of coelenterazine to produce light emission at 482 nm. In vivo an energy transfer to green fluorescent protein occurs and light is emitted at 509 nm. Cloning of the Renilla luciferase gene [267] allowed its use as a reporter gene. Its suitability for this use is confirmed by the several works in the recent literature on this topic. In one of these papers the characteristics of Renilla luciferase were combined with those for GFP through the engineering of a new protein [268]. This bifunctional polypeptide may become a useful tool based on fluorescence for identification of transformed cells at the single-cell level. Simultaneously, it may allow quantifying of promoter activation in transformed tissues and transgenic organisms by measurement of luciferase activity. More recently Renilla luciferase gene was associated with the firefly one to develop dual-luciferase reporter systems. The assay was used to measure translation-coupling efficiency of recording mechanisms such as frame shifting or readthrough [269]. In another work a dual-lucifer- ase assay was employed to simultaneously screen agonist activity at two G-pro- tein-coupled receptors in a 96-well format, resulting in significant time and cost savings [270]. Recently the use as reporter of gene expression in living cells has been extended to the chloroplast genome [271].

5. CONCLUSIONS

The wide collection of analytical applications of the main bioluminescent systems here reported, even necessarily incomplete, is supposedly exhaustive enough to