Chemiluminescence in Analytical Chemistry
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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 |
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2. |
ANALYTICAL APPLICATIONS OF THE MAIN |
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BIOLUMINESCENT SYSTEMS |
251 |
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2.1 |
Firefly Luciferase |
251 |
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2.2 |
Bacterial Luminescence |
261 |
3. |
IMMOBILIZED BIOLUMINESCENT SYSTEMS |
266 |
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4. |
OTHER BIOLUMINESCENT SYSTEMS |
270 |
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4.1 |
Aequorea victoria |
271 |
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4.2 |
Obelin |
274 |
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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-
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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 |
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Table 1 Different Kinds of Bioluminescent Organisms |
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Bioluminescence |
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max range, |
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Type and examples |
in vivo λB (nm) |
Essential factors |
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Bacteria |
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Photobacterium, Vibrio fi- |
478–505 |
Luciferine (an aldehyde), lucif- |
scheri and Vibrio harveyi |
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erase FMNH2 and O2 |
Protozoans |
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Dinoflagellates: Gonyaulux, |
470 |
Luciferine (a biliar pigment), lu- |
Pyrocystis |
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ciferase and O2 |
Coelenterates |
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Hydrozoan jellyfish |
508 |
Photoproteins, Ca2 , a green |
(Aequorea) |
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fluorescent protein |
Anthozoans: Renilla, Ptilo- |
509 |
Luciferase, green fluorescent |
sarcus |
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protein, O2 |
Annelids |
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Polychaetes: Chaetopterus |
465 |
Photoprotein, Fe2 , H2 O2, O2 |
Oligochetes (earthworms) |
500 |
Luciferine (an aldehyde), lucif- |
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erase, H2 O2 |
Molluscs |
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Limpet: Latia |
535 |
Luciferine, luciferase, purple |
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protein, O2 |
Bivalve: Pholas |
490 |
Luciferine (a protein) lucifer- |
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ase, (or Fe2 ), O2 |
Squids: Encleoteuthis, Chiro- |
416–540 |
Luciferine (coelenterazine), lu- |
teutis |
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ciferase, O2 |
Crustacea (Cypridina) |
465 |
Luciferine (Cypridina luciferin), |
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luciferase, O2 |
Shrimps and decapods |
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Oplophorus, Sergestes |
462 |
Luciferine (coelenterazine), lu- |
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ciferase, O2 |
Millipedes: Liminodesmus |
496 |
Photoproteins, ATP, Mg2 , O2 |
Insects: |
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Firefly (Lampiridae): Photi- |
496 |
Luciferine, luciferase, ATP, |
nus, Photuris and others |
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Mg2 , O2 |
Emicordates |
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Balanoglossus |
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Luciferine, luciferase, (Peroxi- |
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dase), H2 O2 |
Fish |
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Batracoides: Porichthys |
459 |
Luciferine (Cypridina luciferin), |
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luciferase, O2 |
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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) |
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Bacteria |
Luciferase (76,000; α,β) |
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Lumazine protein (21,200) |
Dinoflagellates |
Luciferase (130,200) |
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Luciferin-binding protein |
Anthozoa |
Luciferase (35,000) |
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Green-fluorescent protein (52,000; α2) |
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Luciferin-binding protein |
Firefly |
Luciferase (60,000) |
Crustaceans: Cypridina |
Luciferase (68,000; α6) |
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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 |
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Topic |
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Ref. |
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General |
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Chemiluminescence and bioluminescence |
9 |
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Bioluminescence and chemiluminescence |
10 |
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Analytical luminescence: its potential in the clinical laboratory |
11 |
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ATP determination with firefly luciferase |
3 |
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Chemiluminescent and bioluminescent methods in analytical chemistry |
12 |
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Luminometry |
13 |
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Biological diversity, chemical mechanism, and the evolutionary origins of bioluminescent systems |
14 |
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Evolutionary origins of bacterial bioluminescence |
15 |
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Bioluminescence and chemiluminescence, Part B |
1 |
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Fluorescence and bioluminescence measurement of cytoplasmic free calcium |
16 |
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Chemiluminescence: principles and applications in biology and medicine, several chapters on biolum- |
7 |
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inescence |
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Clinical and biochemical applications of luciferase and luciferins |
17 |
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Bioluminescence and chemiluminescence-based fiberoptic sensors |
18 |
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Bioluminescence/chemiluminescence-based sensors |
19 |
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Genetics of bacterial bioluminescence |
4 |
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Chemistries and colors of bioluminescent reactions—a review |
6 |
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Chemiluminescence and bioluminescence |
2 |
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Luminescent techniques applied to bioanalysis |
20 |
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Bioluminescence |
5 |
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Immunoassay, nucleic acid, and reporter gene assays |
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Immunoassay |
21 |
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Bioluminescent immunoassay and nucleic acid assay |
22 |
252
.al et Girotti
Bioluminescence: molecular biology and application |
23 |
Chemiluminescent and bioluminescent reporter gene assays |
24 |
Luciferase and recombinant luciferase labels |
25 |
Symposia |
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Bioluminescence and chemiluminescence, 5th International Symposium on Bioluminescence and |
26 |
Chemiluminescence, 1988: studies and applications in biology and medicine |
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Bioluminescence and chemiluminescence: status report, 7th International Symposium on Biolumines- |
27 |
cence and Chemiluminescence, 1993 |
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Bioluminescence and chemiluminescence: fundamentals and applied aspects, 8th International Sympo- |
28 |
sium on Bioluminescence and Chemiluminescence, 1994 |
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Bioluminescence and chemiluminescence: molecular reporting with photons, 9th International Sympo- |
29 |
sium on Bioluminescence and Chemiluminescence, 1996 |
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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 |
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Literature surveys |
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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 |
Chemistry Analytical in Bioluminescence
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254
Table 3 |
Continued |
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Topic |
Ref. |
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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 |
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Creatures that glow: a book about bioluminescent animals |
55 |
Animals that glow |
56 |
Web sites on bioluminescence |
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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/ |
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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 |
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David Attenborough, ‘‘Talking to Strangers’’ a program in the Trials of Life series |
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The last 15 min of this video talks about bioluminescence |
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.al et Girotti
Bioluminescence in Analytical Chemistry |
255 |
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