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Importance Of Chemiluminescence And Bioluminescence

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Though light is a form of energy, to create light, another form of energy must be supplied for which there are two common ways to occur: incandescence and luminescence. Incandescence is light from heat energy that is from hot objects. If we heat something to a high enough temperature, it will begin to glow. For example, when an electric stove’s heater or metal in a flame begins to glow “red hot”, they exhibit incandescence. Similarly when the tungsten filament of an ordinary incandescent light bulb is heated still hotter, it glows brightly “white hot” by the same means. So much so the sun and stars glow by incandescence.

Contrary to incandescence, luminescence is “cold light” that can be emitted at normal and lower temperatures. In luminescence, some energy source kicks an electron of an atom out of its lowest energy “ground” state into a higher energy “excited” state; then the electron returns the energy in the form of light so it can fall back to its “ground” state. With few exceptions, the excitation energy is always greater than the energy (wavelength, color) of the emitted light.

If we lift a rock, our muscles will be supplying energy to raise the rock to a higher-energy position. If we then drop the rock, the energy we supply is released, of course, some of it in the form of sound, as it drops back to its original low-energy position. With electrical attraction replacing gravity, the atomic nucleus replacing the earth, an electron replacing the rock, and light replacing the sound, it is somewhat the same that happens with luminescence. There are several varieties of luminescence, each named according to the source of energy, or the trigger for the luminescence:

Fluorescence and Photoluminescence are luminescence where the energy is supplied by electromagnetic radiation (rays such as light). Photoluminescence is generally taken to mean “luminescence from any electromagnetic radiation”, while fluorescence is often used only for luminescence caused by ultraviolet, although it may also be used for other photoluminescences. Fluorescence is seen in fluorescent lights, amusement park and movie special effects, the redness of rubies in sunlight, “day-glo” or “neon” colors, and in emission nebulae seen with telescopes in the night sky. Bleaches enhance their whitening power with a white fluorescent material.

Photoluminescence should not be confused with reflection, refraction, or scattering of light, which cause most of the colors we see in daylight or bright artificial lighting. Photoluminescence is distinguished in that the light is absorbed for a significant time, and generally produces light of a frequency that is lower than, but otherwise independent of, the frequency of the absorbed light.

Chemiluminescence is luminescence where the energy is supplied by chemical reactions. The ‘glow-in-the-dark’ plastic tubes used or sold in amusement parks are examples of chemiluminescence. Bioluminescence is luminescence caused by chemical reactions in living things; it is a form of chemiluminescence. Fireflies glow by bioluminescence. Electroluminescence is luminescence caused by electric current. Cathodoluminescence is electroluminescence caused by electron beams; this is how television pictures are formed on a CRT (Cathode Ray Tube). Other examples of electroluminescence are neon lights, the auroras, and lightning flashes. This should not be mistaken for what occurs with the ordinary incandescent electric lights, in which the electricity is used to produce heat, and it is the heat that in turn produces light.

Radioluminescence is luminescence caused by nuclear radiation. Older ‘glow-in-the-dark’ clock dials often used paint with a radioactive material (typically a radium compound) and a radioluminescent material. The term may be used to refer to luminescence caused by X-rays, also called photoluminescence. Phosphorescence is delayed luminescence or “afterglow”. When an electron is kicked into a high-energy state, it may get trapped there for some time (as if we lifted that rock, then set it on a table). In some cases, the electrons escape the trap in time; in other cases they remain trapped until some trigger gets them unstuck (like the rock will remain on the table until something bumps it). Many glow-in-the-dark products, especially toys for children, involve substances that receive energy from light, and emit the energy again as light later.

Triboluminescence is phosphorescence that is triggered by mechanical action or electroluminescence excited by electricity generated by mechanical action. Some minerals glow when hit or scratched, as we can see by banging two quartz pebbles together in the dark. The visible light emitted is often a secondary fluorescence effect, from electroluminescence in the ultraviolet. Thermoluminescence is phosphorescence triggered by temperatures above a certain threshold. This should not be confused with incandescence, which occurs at higher temperatures. In thermoluminescence, heat is not the primary source of the energy; it is rather only the trigger for the release of energy that originally came from another source. It may be that all phosphorescences have a minimum temperature, but many have a minimum triggering temperature below normal temperatures and are not normally thought of as thermoluminescences. Optically stimulated luminescence is phosphorescence triggered by visible light or infrared. In this case red or infrared light is only a trigger for release of previously stored energy.

When two molecules react chemically so that there is a release of energy, that energy sometimes manifests itself not as heat but as light. This occurs because the energy excites the product molecules into which it has been funneled. A molecule in this excited state either relaxes to the ground state, with the direct emission of light, or transfers its energy to a second molecule, which becomes the light emitter. This process is referred to as chemiluminescence. The originally green, now multicolored, commercially made “light sticks” (often in the form of bracelets and necklaces) work in this way, utilizing the (exothermic) reaction of hydrogen peroxide with an oxalate ester. This oxidation reaction produces two molecules of carbon dioxide (CO2), and the released energy is transferred to a fluorescent dye molecule, usually an anthracene derivative. Light sticks were developed by the U.S. Navy as an inconspicuous and easily shielded illumination tool for special operations forces dropped behind enemy lines. Besides their use as children’s toys, they are also used extensively as a navigation aid by divers searching in muddy water. The light sticks glow as a result of the energy released by a chemical reaction.

Chemiluminescence is also found in fireflies. The male firefly uses the reaction of a luciferin substrate and the enzyme luciferase with oxygen, with adenosine triphosphate (ATP) as an energy source, to create the illumination it uses to attract a mate. Because the detection of very minute amounts of light is possible, chemiluminescence and bioluminescence have become the basis of many sensitive analytical and bioanalytical techniques or assays used to quantify particular compounds in samples. Indeed, the use of these techniques is broad enough to justify the existence of a journal devoted to them, the Journal of Bioluminescence and Chemiluminescence.

In 1669 Hennig Brand, a German alchemist, was attempting to recover, by means of intense heat, the gold he hoped was lurking in human urine. The waxy white substance that he did retrieve, which glowed green when exposed to air, was in fact elemental phosphorus. The emission of light observed by Brand was actually chemiluminescence. The light arises from PO2 molecules in an excited state. This excited state of PO2 is brought about by the reaction between PO and ozone, which are both intermediates in the fundamental reaction between oxygen in air and P4 vapor evaporating from the solid white phosphorus. It is unfortunate that the chemiluminescent glow of phosphorus gave rise to the term “phosphorescence.” Scientifically, phosphorescence is a process whereby absorbed photons are emitted at a later time, as exemplified by the glow of a watch face in the dark after its earlier exposure to light.

Luminol (3-aminophthalhydrazide) is used in a commercially available portable device called the Luminox that measures minute concentrations (parts per billion) of the pollutant nitrogen dioxide in air. Luminol is also used frequently in laboratory demonstrations of the chemiluminescence phenomenon. Luminol-mediated chemiluminescence is the result of an oxidation reaction. The oxidation proceeds in two steps, which ultimately lead to the production of the aminophthalate anion in an excited state and the elimination of water and molecular nitrogen. The formation of the strong triple bond (N≡N) is a major factor in the release of energy in the form of light. Probably the simplest chemiluminescent reaction (and one that has been studied extensively) is the reaction between nitric oxide, NO, and ozone, O3. The reaction (with about 10% efficiency) yields nitrogen dioxide in an excited state (NO2*)

NO + O3 = NO2* + O2 and NO2* = NO2 + h ν

This reaction was developed in the early 1970s as a specific and instantaneous method to detect nitric oxide in the exhaust of automobiles. This use of chemiluminescence rapidly led to application of the same phenomenon to monitor the presence of NO in the atmosphere. Both applications continue in use even today. Ozone can easily be produced by passing dry air or oxygen through an electric discharge. The ozone-containing stream and the sample to be evaluated are mixed in a dark chamber adjacent to a photomultiplier tube, and the chemiluminescence signal that is produced is amplified. These devices are capable of monitoring NO levels ranging from parts per trillion to thousands of parts per million; an individual instrument can sometimes measure concentrations extending across six orders of magnitude.

The familiar yellow glow from a natural gas or wood-burning flame is not the result of chemiluminescence, but is due to bright, red-hot particles of carbon soot. The blue, green, and other colors produced when metals are put into flame can indeed be ascribed to chemiluminescence; in these instances the luminescence is accompanied by heat production. It has been found that more than 90 percent of organisms living in the oceans at depths from 200 to 1,000 meters use chemiluminescence for activities such as attracting prey and avoiding predators. Light from the sky is quite weak at those depths; a fish that emits a dim glow from its lower parts could become invisible from below, while a fish without this capability would appear as a dark shadow.

Bioluminescence is the emission of visible light by biological systems, which arises from enzyme-catalyzed chemical reactions. Bioluminescence can be distinguished from chemiluminescence in that it occurs in living organisms and requires an enzyme catalyst. These chemical-dependent emissions of light differ from fluorescence and phosphorescence, which involve the absorption of light by a compound followed by emission of light at a lower energy (higher wavelength) from the excited state of the molecule. The excited molecule produced during bioluminescence reactions, however, is analogous to that produced during fluorescence, and consequently the luminescence emission spectrum can often be related to the fluorescence emission spectrum. It should also be noted that the processes of fluorescence and phosphorescence also occur in living organisms and should not be confused with bioluminescence. The jellyfish is among many bioluminescent species.

Bioluminescence has been observed in many organisms and phyla throughout the terrestrial and aquatic worlds, with the majority of luminescent organisms being found in the ocean. Because of the ease with which light can be detected, recorded observations of bioluminescence extend back several thousand years. Both the ancient Chinese and the ancient Greeks recorded luminescence sightings. Aristotle, in the fourth century B.C.E., wrote that “some things, though they are not in their nature fire, nor any species of fire, yet seem to produce light.”

Luminescent species are found among marine and terrestrial bacteria, annelids or segmented worms, beetles, algae, crustaceans, mollusks, coelenterates, bony fish, and cartilaginous fish. Luminescent vertebrates (except for certain fish), mammals, higher plants, and viruses do not exist—except for those versions created by recombinant technology. Most, if not all, bioluminescence reactions have oxygen as a common reactant and a conjugated system as part of one of the substrates—both needed to generate molecules in an excited state, leading to the emission of light in the visible region. However, the bioluminescence reactions in some organisms are quite different from those in other organisms, and consequently the enzymes catalyzing the reactions (luciferases) and the substrates (often but not always referred to as luciferins) are also quite distinct. Four bioluminescence systems (fireflies, dinoflagellates, bacteria, and imidazolopyrazine-based e. g., coelenterates) have been studied in greatest detail, and their chemical reactions reflect both their differences and their common features.

Luciferases from click beetles, fireflies, and railway worms catalyze the ATP-dependent decarboxylation of luciferin. An AMP derivative of luciferin is formed, which subsequently reacts with O2. Cleavage of this dioxy derivative results in the emission of light characterized by wavelengths ranging from 550 nanometers ( green) to 630 nanometers (red, depending on the particular luciferase), and the release of CO2. Fireflies generally emit in the yellow to green range, as part of a courtship process; click beetles emit green to orange light; whereas railway worms emit red light, with green light being emitted on movement.

Much of the brightness that is observed on the surface of the oceans is due to the bioluminescence of certain species of dinoflagellates, or unicellular algae, and this bioluminescence accounts for many of the recorded observations that have described the apparent “phosphorescence” of the sea. Dinoflagellates are very sensitive to motion induced by ships or fish, and respond with rapid and brilliant flashes, thus causing the glow that is sometimes seen in the wake of a ship. The luciferin in these instances is a tetrapyrrole containing four five-member rings of one nitrogen and four carbons, and its oxidation, catalyzed by dinoflagellate luciferase, results in blue-green light centered at about 470 nanometers.

Bacterial luciferase catalyzes the reaction of reduced flavin mononucleotide (FMNH2) with O2 to form a 4a-peroxyflavin derivative that reacts with a long chain aldehyde leading to the emission of blue-green light (490 nanometers) and the formation of riboflavin phosphate (FMN; the phosphorylated form of vitamin B2), H2O, and the corresponding fatty acid. Luminescent bacteria are found throughout the marine environment, living free, in symbiosis, or in the gut of marine organisms (including many fish and squid), as well as in the terrestrial environment as symbionts of nematodes.

The luciferins believed to be the most widespread among phyla living in the ocean have structures based on imidazolopyrazine, for example, coelenterazine, found in luminescent coelenterates contains imidazolopyrazine as its central bicyclic ring. The typical reaction involves the oxidation of the imidazolopyrazine ring with the emission of blue light (460–480 nanometers), and proceeds according to a mechanism that is very similar to that of the oxidation of firefly luciferin. Among the most commonly studied imidazolopyrazine-utilizing organisms are species of Renilla (sea pansy) and Aequorea (jellyfish) both of which utilize coelenterazine. The luciferin of a crustacean (Cypridina or Vargula) also is an imidazolopyrazine-based compound related to coelenterazine. The luciferases of the luminescent species, however, vary widely. Recent evidence suggests that some, and possibly many, marine luminescent organisms (including the jellyfish) acquire luciferins via the ingestion of other luminescent organisms, which would account for the widespread distribution of imidazolopyrazine-based luciferins. Many luminescent species also have a binding protein that releases the luciferin upon Ca2+ uptake, while some have a fluorescence protein that absorbs and then emits light at a higher wavelength.

Although other luminescent systems have been studied (including those of the fireworm and the limpet, both of which use aldehydes as luciferins), bioluminescence remains somewhat mysterious. Elucidation of the chemical and biological bases for luminescence systems in other organisms should improve understanding of why the remarkable and beautiful phenomenon of bioluminescence appears in so many species.

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