Chemiluminescence in Analytical Chemistry

246

Knight

72.AF Martin, TA Nieman. Biosens Bioelectron 12:479–489, 1997.

73.Boehringer Mannheim GmbH, Mannheim. Germany. Company literature.

74.H Yu, PJ Stopa. In: Am Chem Soc Symp Ser, Vol 646, Environmental Immunochemical Methods, 1996, pp 297–306.

75.R Wilson, MH Barker, DJ Schiffrin, R Abuknesha. Biosens Bioelectron 12:277– 286, 1997.

76.J Kankare, K Haapakka, S Kulmala, V Na¨nto¨, J Eskola, H Takalo. Anal Chim Acta 266:205–212, 1992.

77.X-H Xu, AJ Bard. J Am Chem Soc 117:2627–2631, 1995.

78.SJ Haswell. Analyst 122:R1–R10, 1997.

79.J Kremesko¨tter, R Wilson, DJ Schiffrin, BJ Luff, JS Wilkinson. Measurement Sci Technol 6:1325–1328, 1995.

80.Y-T Hsueh, RL Smith, MA Northrup. Sens Actuat B Chem 33:110–114, 1996.

81.GC Fiaccabrino, NF deRooij, M Koudelka-Hep. Anal Chim Acta 359:263–267, 1998.

82.JE Vitt, RC Engstom. Anal Chem 69:1070–1076, 1997.

83.AV Kukoba, NN Rozhitskii. Russian Electrochem 29:345–351, 1993.

84.RC Engstrom, PL Nohr, JE Vitt. Colloids Surfaces A: Physicochem Engin Asp 93: 221–227, 1994.

85.P Hopper, WG Kuhr. Anal Chem 66:1996–2004, 1994.

10

Applications of Bioluminescence in Analytical Chemistry

Stefano Girotti, Elida Nora Ferri, Luca Bolelli, Gloria Sermasi, and Fabiana Fini

University of Bologna, Bologna, Italy

1.	AN INTRODUCTION TO BIOLUMINESCENCE		247
2.	ANALYTICAL APPLICATIONS OF THE MAIN
	BIOLUMINESCENT SYSTEMS		251
	2.1	Firefly Luciferase	251
	2.2	Bacterial Luminescence	261
3.	IMMOBILIZED BIOLUMINESCENT SYSTEMS		266
4.	OTHER BIOLUMINESCENT SYSTEMS		270
	4.1	Aequorea victoria	271
	4.2	Obelin	274
	4.3	Renilla Luciferase	275
5.	CONCLUSIONS		275

1. AN INTRODUCTION TO BIOLUMINESCENCE

The emission of light after chemical excitation is called chemiluminescence (CL). If it occurs in biological systems it is known as bioluminescence (BL) or, de-

247

248	Girotti et al.

scribed in a more detailed manner, BL is a case of enzyme-catalyzed CL (Fig. 1) [1, 2].

For most people, BL is represented by the flash of the firefly or the ‘‘phosphorescence’’ that frequently occurs on agitating the surface of ocean water. Chemical excitation, luminescent reactions occurs in almost all zoological kingdoms (bacteria, dinoflagelates, crustacea, worms, clams, insects, and fishes) except higher vertebrates: BL is not found in any organisms higher than fish. In most cases this phenomenon occurs within specialized cells called photocytes [3–5]. As shown in Table 1, BL occurs in many terrestrial forms but is most common in the sea, particularly in the deep ocean, where the majority of species are luminescent [6].

BL has independently evolved many times; some 30 different independent systems are still existent [7]. Thus the responsible genes are unrelated in the various organisms, the enzymes show no homology to each other, and the substrates are chemically unrelated. There is, however, one common thread tying different systems at molecular level. All involve exergonic reactions of molecular

Figure 1 Scheme of chemiluminescent and bioluminescent light emission.

Bioluminescence in Analytical Chemistry		249
Table 1 Different Kinds of Bioluminescent Organisms
	Bioluminescence
	max range,
Type and examples	in vivo λB (nm)	Essential factors
Bacteria
Photobacterium, Vibrio fi-	478–505	Luciferine (an aldehyde), lucif-
scheri and Vibrio harveyi		erase FMNH2 and O2
Protozoans
Dinoflagellates: Gonyaulux,	470	Luciferine (a biliar pigment), lu-
Pyrocystis		ciferase and O2
Coelenterates
Hydrozoan jellyfish	508	Photoproteins, Ca2 , a green
(Aequorea)		fluorescent protein
Anthozoans: Renilla, Ptilo-	509	Luciferase, green fluorescent
sarcus		protein, O2
Annelids
Polychaetes: Chaetopterus	465	Photoprotein, Fe2 , H2 O2, O2
Oligochetes (earthworms)	500	Luciferine (an aldehyde), lucif-
		erase, H2 O2
Molluscs
Limpet: Latia	535	Luciferine, luciferase, purple
		protein, O2
Bivalve: Pholas	490	Luciferine (a protein) lucifer-
		ase, (or Fe2 ), O2
Squids: Encleoteuthis, Chiro-	416–540	Luciferine (coelenterazine), lu-
teutis		ciferase, O2
Crustacea (Cypridina)	465	Luciferine (Cypridina luciferin),
		luciferase, O2
Shrimps and decapods
Oplophorus, Sergestes	462	Luciferine (coelenterazine), lu-
		ciferase, O2
Millipedes: Liminodesmus	496	Photoproteins, ATP, Mg2 , O2
Insects:
Firefly (Lampiridae): Photi-	496	Luciferine, luciferase, ATP,
nus, Photuris and others		Mg2 , O2
Emicordates
Balanoglossus		Luciferine, luciferase, (Peroxi-
		dase), H2 O2
Fish
Batracoides: Porichthys	459	Luciferine (Cypridina luciferin),
		luciferase, O2

250	Girotti et al.

oxygen with different substrates (luciferins) catalyzed by enzymes (luciferases), resulting in photons of light ( 50 kcal). Luciferase and luciferin are generic terms referring to an enzyme that catalyzes the oxidation of a substrate, such as luciferin, and to a reduced compound that can be oxidized in an appropriate environment to produce an electronically excited singlet state. Light is produced on its return to the ground state. All involve a luciferase-bound peroxy-luciferin intermediate, the breakdown of which provides energy for excitation. Under in vitro conditions the quantum yield of such reactions can be as high as 0.9. In Table 2 the properties of some bioluminescent systems are listed. The maximum wavelength of the light emitted is often in the range 460–560 nm; then the color ranges from the red of worm through the deep blue characteristic of most marine creatures. Several factors affect the color of BL [6]. In the simplest case, the emission matches the fluorescence of an excited luciferase-bound product of the reaction. The luciferase structure can itself alter the color, as in the firefly, where single amino acid substitutions result in significant shifts in the emission spectrum. In bacteria and coelenterates, the chromophores of accessory proteins associated with luciferases may serve as alternate emitters, such as the yellow fluorescent protein (YFP) in bacteria and the green fluorescent protein (GFP) in coelenterates, now used as reporter genes and cellular markers [8]. The cell biology and regulation of BL differ among groups. While bacteria and some other systems emit light continuously, in many the luminescence occurs as flashes, typically of 0.1–1 s duration.

BL research has increasingly drawn scientists’ renewed attention during the last years. One of the main reasons for this concern is due to the development of gene technology and the applications of its many new methods to study BL at the molecular level. Progress in the fundamental knowledge of BL has led to numerous gene-reporting techniques, thanks to which new basic knowledge is

Table 2 Properties of Some Bioluminescent Systems

Organism	Protein involved (Mass-Da, subunits)
Bacteria	Luciferase (76,000; α,β)
	Lumazine protein (21,200)
Dinoflagellates	Luciferase (130,200)
	Luciferin-binding protein
Anthozoa	Luciferase (35,000)
	Green-fluorescent protein (52,000; α2)
	Luciferin-binding protein
Firefly	Luciferase (60,000)
Crustaceans: Cypridina	Luciferase (68,000; α6)

Bioluminescence in Analytical Chemistry

251

now acquired in many areas of biology and medicine. As a consequence, there is a growing interest and demand of research institutions other than biomedical to utilize highly sensitive bioluminescent techniques for analytical purposes.

This review deals mainly with BL analytical applications in the last 10– 15 past years, but some previous fundamental works are also listed. In Table 3 some fundamentals references of general interest and the findings of recent symposia on this topic are collected. In the journal Luminescence, the Journal of Biological and Chemical Luminescence (previously Journal of Bioluminescence and Chemiluminescence) are also reported surveys of the recent literature on selected topics (like ATP or GFP applications), instruments, and kits commercially available.

2.ANALYTICAL APPLICATIONS OF THE MAIN BIOLUMINESCENT SYSTEMS

2.1 Firefly Luciferase

The most popular system in mechanistic and model studies as well as in analytical applications (clinical, food, environmental) appears to be that of firefly luciferin and luciferin-type-related model luminescence [3, 5, 23, 57]. The luciferase from Photinus pyralis, Photinus luciferin 4-monooxygenase (ATP-hydrolyzing), EC 1.13, 12.7, is a hydrophobic enzyme that catalyzes the air oxidation of luciferin in the presence of ATP and magnesium ions to yield light emission:

Luciferase, Mg2

ATP reduced luciferin O2 → AMP oxyluciferine hν

The mechanism is more complex than reported above, and starting with the pioneer studies of DeLuca and McElroy [58], it has been the object of deep investigations. A more detailed scheme is reported in Figure 2. ATP is consumed as a substrate and photons at a wavelength of 562 nm are emitted. The quantum yield of this reaction is 0.9 einstein mol/L of luciferin. Considering the stoichiometry of the reaction for one ATP molecule consumed, approximately one photon is emitted. This property, together with the high nucleoside specificity of the enzyme, makes this reaction an ideal analytical system for assaying ATP presence, ATP production or consumption in dependence of enzymatic activity, and for quantification of substrates linked to the ATP metabolism. ATP is the most important and central coupling agent between exergonic and endergonic processes and it is ubiquitous in living organisms where it functions as an allosteric effector, as a group-carrier coenzyme, and as a substrate. Because of the essentiality of ATP and of the related enzymes and substrates in metabolism, accurate, sensitive,

Table 3	Recently Reviewed Analytical Applications of Bioluminescent Systems
Topic		Ref.
General
Chemiluminescence and bioluminescence		9
Bioluminescence and chemiluminescence		10
Analytical luminescence: its potential in the clinical laboratory		11
ATP determination with firefly luciferase		3
Chemiluminescent and bioluminescent methods in analytical chemistry		12
Luminometry		13
Biological diversity, chemical mechanism, and the evolutionary origins of bioluminescent systems		14
Evolutionary origins of bacterial bioluminescence		15
Bioluminescence and chemiluminescence, Part B		1
Fluorescence and bioluminescence measurement of cytoplasmic free calcium		16
Chemiluminescence: principles and applications in biology and medicine, several chapters on biolum-		7
inescence
Clinical and biochemical applications of luciferase and luciferins		17
Bioluminescence and chemiluminescence-based fiberoptic sensors		18
Bioluminescence/chemiluminescence-based sensors		19
Genetics of bacterial bioluminescence		4
Chemistries and colors of bioluminescent reactions—a review		6
Chemiluminescence and bioluminescence		2
Luminescent techniques applied to bioanalysis		20
Bioluminescence		5
Immunoassay, nucleic acid, and reporter gene assays
Immunoassay		21
Bioluminescent immunoassay and nucleic acid assay		22

252

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Bioluminescence: molecular biology and application	23
Chemiluminescent and bioluminescent reporter gene assays	24
Luciferase and recombinant luciferase labels	25
Symposia
Bioluminescence and chemiluminescence, 5th International Symposium on Bioluminescence and	26
Chemiluminescence, 1988: studies and applications in biology and medicine
Bioluminescence and chemiluminescence: status report, 7th International Symposium on Biolumines-	27
cence and Chemiluminescence, 1993
Bioluminescence and chemiluminescence: fundamentals and applied aspects, 8th International Sympo-	28
sium on Bioluminescence and Chemiluminescence, 1994
Bioluminescence and chemiluminescence: molecular reporting with photons, 9th International Sympo-	29
sium on Bioluminescence and Chemiluminescence, 1996
Industrial application of bioluminescent ATP assay, ATP96	30
Bioluminescence and chemiluminescence: perspectives for the 21st century, 10th International Sympo-	31
sium on Bioluminescence and Chemiluminescence, 1998
Literature surveys
Bioluminescence and chemiluminescence, 1989	32
Bioluminescence and chemiluminescence, Russian literature	33
Bioluminescence and chemiluminescence, 1990 part 1	34
Bioluminescence and chemiluminescence, 1990 part 2	35
Bioluminescence and chemiluminescence, 1991 part 1	36
Bioluminescence and chemiluminescence, 1991 part 2	37
Nucleic acid hybridization assays	38
Immunoassay and protein blotting assays	39
Bioluminescence and chemiluminescence, 1992	40
Luciferase reporter genes—lux and luc	41
Bioluminescence and chemiluminescence, 1993	42
Bioluminescence and chemiluminescence, 1994 part 1	43

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254

Table 3	Continued

Topic	Ref.
Bioluminescence and chemiluminescence, 1994 part 2	44
Bioluminescence and chemiluminescence, 1994 part 3	45
Bioluminescence and chemiluminescence, 1995 part 1	46
Bioluminescence and chemiluminescence, 1995 part 2	47
Bioluminescence and chemiluminescence, 1995 part 3	48
Green fluorescent protein	49
Bioluminescence and chemiluminescence, 1996	50
Bioluminescence and chemiluminescence, 1997 part 1	51
Bioluminescence and chemiluminescence, 1997 part 2	52
Commercial available luminometers, imaging devices, and reagents, survey update 5	53
Commercial available luminometers, fluorometers, imaging devices, and reagents, survey update 6	54
Teaching
Creatures that glow: a book about bioluminescent animals	55
Animals that glow	56
Web sites on bioluminescence
The bioluminescence web page	http://lifesci.ucsb.edu/ biolum/
Scripps Institution of Oceanography	http://siobiolum.ucsd.edu/Biolum_intro.html
Bioluminescence: a proctor project by R Abaza	http://www.biology.lsa.umich.edu/ www/
	bio311/projects/ronney/biochem.shtml
Bioluminescence and biological fluorescence	http://www.herper.com/Bioluminescence.html
Bioluminescence studies research and resources by John E. Wampler (Renilla green fluorescent pro-	http://bmbiris.bmb.uga.edu/wampler/biolum/
tein and other organisms)	index.html#web
Video
David Attenborough, ‘‘Talking to Strangers’’ a program in the Trials of Life series
The last 15 min of this video talks about bioluminescence

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Figure 2 A more detailed scheme of ATP firefly luciferase reaction. LF luciferase; OL oxiluciferine; LFH2 reduced luciferase; PP pirophosphate; OL* oxiluciferine in the excited state.

and simple methods are required for its determination. These methods are applicable to medicine, biology, environmental studies, agriculture, and industry, as well as in the laboratory, pointing out the great versatility of the system. Some of the numerous applications of ATP bioluminescent assay are listed in Table 4.

One problem with the ATP assay in aqueous media is that the enzyme requires hydrophobic media; the reaction rate of luciferase-catalyzed reactions is variously affected by the presence of detergents [117, 118]. The presence of cationic liposomes improves sensitivity by a factor of five times compared to that in water alone [119].

Another characteristic to take into account is that ATP is an endogenous component of the cells, both somatic and bacterial. Therefore, an extraction step must to be included in the assay protocol; it is very simple and quick to perform. Several extraction methods have been reported, both physical and chemical, such as heating and the use of surfactants, trichloroacetic acid, and organic solvents [89, 120, 121]. The chemical methods are generally preferred; the addition of a surfactant can be effective in most cases. The use of mild or strong extraction