1274 Analyst November 1983 Vol. 108 p p . 1274-1296 Chemiluminescent and Bioluminescent Methods in Analytical Chemistry A Review Larry J. Kricka and Gary H. G. Thorpe Department of Clinical Chemistry University of Birmingham and Department of Clinical Chemistry Wolfson Research Laboratories Queen Elizabeth Medical Centre Birmingham B 15 2TH Summary of Contents Introduction Types of chemiluminescent and bioluminescent reactions Chemiluminescent reactions Acyl hydrazides Imidazoles Acridinium salts Oxalate esters Miscellaneous Firefly Marine bacteria Aequorea Renilla Pholas Instrumentation Reagents Luminescent assays Bioluminescent reactions Advantages and disadvantages Practical considerations Water Buffers Cofactors Auxiliary enzymes Containers Light detection devices Mixing Applications Gas-phase luminescent reactions Luminescent reactions in the liquid phase Assays for metals Calcium Magnesium Zinc Transition metals Enzyme assays Assays for carbohydrates Assays for nucleotides Steroid assays Assays for drugs Chemiluminescent reagents Bioluminescent reagents Immobilised luminescent reagents Immunoassa y Analytical applications of intact bioluminescent organisms Luminescent detectors in chromatography Miscellaneous reactions and assays Conclusions References Keywords Review ; chemiluminescent reactions ; bioluminescent reaction KRICKA AND THORPE 1275 Introduction Luminescence is a generic term that covers a range of processes which produce 1ight.l This review is restricted to the analytical applications of chemiluminescence the luminescence that arises during the course of a chemical reaction and bioluminescence a special type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction.Light is produced when molecules formed in an electronically excited state decay to the ground state. Alternative pathways that lead to the production of product molecules in the ground state or decay of excited-state mole-cules to product in its ground state via radiationless transitions will decrease the light produced in the reaction. Generally the quantum yield i.e. number (or rate) of molecules emitting light divided by the number (or rate) of molecules reacting is in the range 1 4 % for chemiluminescent reactions whereas quantum yields approaching 100% are encountered in the more efficient bioluminescent reactions.Both are examples of luminescent processes but differ in the source of energy producing excited-state molecules. In chemiluminescence it is the energy of a chemical reaction whereas in fluorescence it is incident radiation that produces excited-state molecules. Traditionally chemi- and bioluminescent methods have not played at1 important role in routine analytical chemistry. The availability of suitable commercial instrumentation for light measurement ,2 an improved range and quality of bioluminescent and chemiluminescent reagents and the impetus for research in this area provided by the use of chemi- and bio-luminescence in clinical research may well change this situation.The purpose of this review is to delineate the scope of chemiluminescence and bio-luminescence in analytical chemistry with the emphasis on liquid-phase assays. Chemical and biochemical reactions producing light as well as their mechanisms and applications, have stimulated the reviewing propensity of many so much so that in order to avoid unnecessary and possibly plagiaristic repetition we have dealt very briefly with some aspects of the topic and as appropriate cited previous reviews. In the older literature chemiluminescence has been confused with fluorescence. Types of Chemiluminescent and Bioluminescent Reactions Numerous chemi- and bioluminescent reactions are known although relatively few have A selection of these reactions are described in the following Several less well known or utilised reactions are included because in the future, proved useful analytically.sections. these may provide the basis for new analytical methods. Chemiluminescent Reactions Reactions that produce chemiluminescence are relatively uncommon as most chemical reactions release energy as heat via vibrational excitation of ground-state products. The energy of photons in the visible region (400-560 nm) lies between 44 and 71 kcal mol-l; hence a minimum requirement for chemiluminescence is that the reaction produces 44 kcal mol-l of energy. Free-energy changes of this magnitude are usually encountered in oxidation reactions and thus most chemiluminescent reactions have been discovered amongst oxidation reactions involving oxygen peroxide or other strong oxidant^.^ In most instances the detailed mechanisms of chemiluminescent reactions are not under-stood.From the analytical point of view this is a disadvantage as it is difficult to control or predict interferences when such reactions are used routinely. Nevertheless a number of chemiluminescent reactions are now in regular use. The most popular are reactions based on cyclic acyl hydrazides (e.g. luminol) and acridine derivatives (q. lucigenin). Many other classes of organic and organometallic compounds undergo chemiluminescent reactions and these are described in the following sections in order to illustrate the scope of chemi-luminescent reactions. Acyl hydrazides The chemiluminescen t oxidation reactions of luminol [5-amino-2,3-dihydro-1,4-phthalazine dione (1) 1 were discovered by Albrecht in 1928.6 Luminol undergoes chemiluminescent reactions with a range of oxidants including oxygen hypochlorite iodine permanganate 1276 KRICKA AND THORPE CHEMILUMINESCENT Analyst Vol.108 0 0 1-5 Ph Ph 6 NOB- Me Ph 7 FSO, Me +hv Me I 9 Me NO3-8 CI (?I 0 W0 10 p FLR" + 2C02 hv Fig. 1 . Cheiiiiluniinescent compounds and reactions. Formulae 1-5 are as follows 1 R' = H R2 = NH,; 2 R1= NH, 1x2 = H ; 3 I<' = HO,CCH,COSH(CH,),NC?HJ R2 = H ; 4 K' = H,N(CH2)&JC2H,, R2 = H; and 5 R1 = H,N(CH,),NC2H, R2 = H. peroxide and perborate. For some oxidants e.g. peroxide a co-oxidant or catalyst [e.g., peroxidase haemin Fe(CN),3- Ni2+ Cr3+] is also required for chemiluminescence.For certain catalysts notably Cr(III) Co(II) Fe(I1) and Ni(II) the chemiluminescence is en-hanced by halide ions. In the luminol - Cr(II1) - peroxide reaction enhancement increases in the order Br- > C1- > I;-.' Light emission occurs around 425 nm and the emitter is the excited-state aminophthalate dianion (Fig. 1). The cliemiluminescent reaction is not very efficient (<1%) and the efficiency is further reduced by a shift in the position of the amino group to the 6-position. This substance isoluminol (2) is only about 10% as efficient as the isomeric luminol. Other niodifications such as an increase in the conjugation e.g., structure 6 improved the chemiluminescent efficiency seven-fold.8 Unfortunately such sub-stances are not commercially available.Alkylation of the 5-amino group of luminol decreases chemiluminescence whereas the converse is true for isoluminol. Substitution of the hetero-cyclic ring abolishes clieiiiilumine~cence.~ Recently a number of iso-luminol derivatives have become conimercially available (LKB ll'allac Oy) e.g. (3-5) and the impetus for this has been the use of such compounds as labels in inimunoassays. Provision of a reactiv November 1983 AND BIOLUMINESCENT METHODS. A REVIEW 1277 amino group separated from the basic luminescent structure by a hexamethylene spacer group provides a very convenient site for linking to proteins by any of a number of conjugation reactions (see Immunoassay) . Chemiluminescent oxidation of luminol by peroxide at high pH can be enhanced by the addition of certain organic compounds and up to 25000-fold enhancement of light intensity has been reported.1° The most effective compounds were diazonium salts e.g.p-chloro-benzenediazonium fluoroborate (250 000-fold) and halonitromethanes e.g. tribromonitro-methane (150000-fold enhancement). In all about 47 different enhancers have been described. The mechanism is believed to involve a nucleophilic attack of peroxide on the electrophilic organic compound followed by oxidation of the luminol by the intermediate hydroperoxide. The degree of enhancement seems to be pH dependent and at lower pH, e.g. 8.0 the enhancement is diminished. Monoacyl hydrazides are also chemilumine~cent,~~~ but have not been used as analytical reagents.Imidazoles The chemiluminescent oxidation reaction of lophine [2,4,5-triphenylimidazole (7)] has been known since 1877.l1 Suitable oxidising agents include hydrogen peroxide - sodium hypo-chlorite hydrogen peroxide - potassium hexacyanoferrate(II1) and oxygen. The mechanism for the last oxidant in aqueous alkaline dimethyl sulphoxide is thought to proceed via the formation of a hydroperoxide intermediate which rearranges to form a dioxetane which then decomposes with the emission of light (Fig. 1). The hydroperoxide intermediate has been synthesised and shown to chemiluminesce when exposed to basic ethanol. Like luminol and lucigenin the chemiluminescent oxidation of lophine is catalysed by various ions e.g., AuC14- C10- Mn04- Fe(CN),3- and Cr(III).12913 Acridinium salts Lucigenin [bis-N-methylacridinium nitrate (S)] is the best known example of this class of chemiluminescent compound~.~~J~ It undergoes a chemiluminescent oxidation with peroxide under basic conditions.The emitter is the excited-state product N-methylacridone (Fig. 1). An intermediate dioxetane has been synthesised and shown to decompose with the emission of light. A range of substances will initiate lucigenin chemiluminescence including acetone, hypoxanthine - xanthine oxidase,16 reductants such as hydroxylamine ascorbate creatinine and fructose under alkaline conditions1’ and in the presence of oxygen nucleophiles such as trifluoroethoxide cyanide and eth~1amine.l~ Detailed studies of the kinetics of lucigenin -peroxide and lucigenin - oxygen reactions have been published by Maskiewicz et a1.1*J5 Acridinium phenyl carboxylates e.g.~-acetylphenyl-lO-methylacridinium-9-carboxylate fluorosulphonate (9) also undergo a chemiluminescent oxidation with peroxide. This type of compound is not water soluble but it can be used in aqueous systems if it is dissolved in a water-miscible solvent such as acetonitrile.ls The quantum yield for the lucigenin reaction is low (1.6%). Oxalate esters The chemiluminescent oxidation of certain oxalate esters in the presence of a fluorescer are amongst the most efficient chemiluminescent reactions known (quantum yields up to 27%) and form the basis of the “light sticks” produced by the Cyanamid C ~ m p a n y . ~ ~ ~ ~ ~ A possible reaction mechanism for the oxidation of bis(2,4,6-trichlorophenyl) oxalate (10) by peroxide in the presence of rubrene is presented in Fig.1. The intensity and lifetime of the light emission can be altered by the addition of weak bases (e.g. triethylamine) and weak acids, and the emission wavelength can be varied by changing the fluorophor. A disadvantage of oxalate esters is their insolubility in water. Mixed solvents are required and dioxane - water, tert-butanol - water and ethyl acetate - methanol - water3 have been used successfully as reaction media. Miscellaneous briefly in order to establish the scope of chemiluminescence. Many other substances undergo chemiluminescent reactions and these will be dealt wit 1278 KRICKA AND THORPE CHEMILUMINESCENT Analyst "01. 108 Reactions that involve singlet-state oxygen may produce chemiluminescence.This species is involved or has been implicated in the following chemiluminescent reactions hydrogen peroxide - hypochlorite,21 triphenyl phosphite - ozone,22 hydrogen peroxide - pyrogallol -formaldehyde hydroperoxide and dialkylperoxide decomposit ion.23 Autoxidation reactions are chemiluminescent and a weak chemiluminescence emission has been detected from organic compounds and non-metallic substances (papers plastics cloth) in the presence of atmospheric oxygen. Oxidation of carbenes and Grignard reagents by oxygen nitrites by peroxide iron(II1) compounds by peroxide in the presence of hydrogen carbonate and quinones or sulphite by either oxygen or peroxide is accompanied by emission of light.24 Siloxene a polymer [(S$H,O,),]+ produced from the acidic hydrolysis of calcium silicide emits light when treated with oxidising agents [permanganate nitric acid cerium(1V) ions] and this substance has been advocated as a chemiluminescent indicator for redox titratiom25 The combination of radical anions and radical cations produces excited-state species that decay to the ground state with emission of light.The mutual annihilation of electro-chemically generated diphenylanthracene radical anions and cations and the recombination of thiantlirene radical cations and diphenylanthracene radical anions represent particularly efficient examples of this type of reaction (quantum yields up to 20y0).26 Bioluminescent Reactions Bioluminescence is a widely distributed phenomenon in nature (Table I) and is particularly common amongst marine organisms.Examples of bioluminescent bacteria fungi insects, fishes crustaceans and molluscs are known and an authoritative listing has been compiled by Herring.27 The most extensively studied bioluminescent organisms are the firefly (particularly Photinus +yralis) marine bacteria such as Beneckea harveyi and Photohacteriwn Jischeri the comb jelly Renilla reniformis and the jelly fish Aequorea. Studies of other organisms have been limited by their availability. Some of these contain bioluminescent substances that have considerable analytical potential (e.g. Pholas dactylm) . However their scarcity coupled with the complex nature of the extraction and purification of the active bioluminescent substances have precluded the serious consideration of such substances as reagents for routine analysis.The rapid growth in genetic engineering techniques may lead to a reappraisal of these bioluminescent substances as they could in theory be prepared in large amounts by such techniques. Indeed the in vitro synthesis of sub-units of bacterial luciferase in Escherichia coli has already been accompli~hed.~*~29 TABLE I CLASSIFICATION OF BIOLUMINESCEN'T ORGANISMS2' Type Organisms Bacteria . . . . Photobacterium fischeri Beneckea harveyi Fungi . . Xylaria Armillaria Omphalia Plants . . . . Noctiluca Gonyaulax Pyrodinium Animals . . . . Photinus pyralis A equorea Pelagia Balanoglossus Ptychodera, Renilla reniformis Cyfiridina Pholas dactylus Diplocardia longa, Latia neritoides Arachnocampa luminosa Two terms in common use and which sometimes cause confusion are luciferase and luciferin.Luciferases are enzymes that catalyse the oxidation of a substrate a luciferin with the concomitant emission of light. Luciferases and luciferins from different bioluminescent organisms differ greatly. For example the structures of luciferins from several different types of bio-luminescent organism are shown in Fig. 2. These are generic terms originally coined by Dubois in the 1880~.~* FireJy Firefly luciferase (E.C. 2.8.2.10) catalyses the ATP-dependent oxidation of D-luciferin (LH,). Initially a luciferase luciferyl adenylate (LH, AMP) complex is formed and this reacts with oxygen to produce oxyluciferin (the emitter) carbon dioxide AMP and light. At pH 8.6 the peak emission occurs at 560 nm November 1983 AND BIOLUMINESCENT METHODS.A REVIEW Luciferase + LH + MgATP -+ luciferase LH AMP + pyrophosphate Luciferin LH,AMP + 0 -+ oxyluciferin + CO + AMP + light 1279 However under acidic conditions (pH 7.0) the colour of the emitted light changes from yellow - green to red (peak emission ca. 615 nm). Most photomultiplier tubes are chosen to be maximally sensitive in the yellow - green region so that failure to control the pH or the presence of divalent metal ion impurities (particularly in buffers) can invalidate analytical results obtained using the firefly r e a ~ t i o n . ~ ~ ~ ~ , A similar effect is produced by divalent metal ions such as zinc. A B C D Fig. 2. Luciferins A Cypridinia; B coelenterate; C firefly; and D bacterial.Luciferase is a protein of relative molecular mass 50 000 daltons. Equilibrium dialysis, photon yield and chromatographic studies have shown that the enzyme has one binding site for oxyluciferin and ATP-Mg and two binding sites for ATP AMP and luciferyl adenylate per 100000 daltons of luciferase. These data indicate that either a luciferase dimer con-sisting of identical sub-units or possibly of non-identical sub-units of relative molecular mass 50000 are involved in the light-producing reaction. As yet it has not proved possible to distinguish between these two possibilities. The product oxyluciferin binds tightly to luciferase and this leads to product inhibition. Recently it has been demonstrated that this can be overcome by adding non-ionic detergents at concentrations above their critical micellar concentration^.^^ In the presence of non-ionic detergents such as Tween-20 or Triton X-100 bioluminescence is stimulated.Enhancement of light levels in this way could be valuable especially in firefly reactions producing relatively low light levels. Unfortunately proteins interfere with the mechanism of the detergent action and thus detergent-stimulated bioluminescence may not be widely applicable in analysis. Even the small amounts of protein added as a stabiliser to commercial firefly luciferase preparations abolish the detergent stimulation of light emission. Firefly luciferase is prepared by extraction of firefly tails and purified by crystallisation. The other component of this bioluminescent system luciferin is also contained in the tails but is more conveniently obtained by chemical synthesis.34 Both luciferase and luciferin are readily available from a number of suppliers.Marine bacteria Bioluminescent marine bacteria contain a luciferase that catalyses the oxidation of a long-chain aliphatic aldehyde (RCHO) and reduced flavin mononucleotide (FMNH,) and this reaction is accompanied by the emission of light (peak light emission 490 nm). The bacteri 1280 KRICKA AND THORPE CHEMILUMINESCENT Analyst Vol. 108 also contain an NAD(P)H FMN oxidoreductase which supplies FMNH according to the following equations : NAD(P)H FMN oxidoreductase FMN + NAD(P)H +NAD(P) + FMNH, Lucif erase FMNH + RCHO + 0 - -+ FMN + RC0,H + H,O + light Beneckea harveyi contains both an NADH and an NADPH FMN oxidoreductase and these can be isolated and purified by sequential ion-exchange gel filtration and affinity chromato-gra~hy.~5 In contrast the marine bacteria Photobacterizcm jischeri contains a single mixed specificity oxidoreductase [NAD(P)H FMN oxidoreductase] and currently this is the only oxidoreductase that can be purchased in a purified form (Boehringer Corporation).Luciferase from B. harveyi consists of two non-identical sub-units (relative molecular mass 37000 and 42000 daltons) and this enzyme can be isolated from lysed bacteria using ion-exchange chromatography (DEAE-Sephadex) . Recently a simple affinity chromatographic method using 2,2-diphenylpropylamine-Sepharose has been described and this may prove to be the purification method of choice.36 NADH FMN oxidoreductase is a polypeptide of relative molecular mass 23000 daltons.In comparison with luciferase the amount of this enzyme extractable from the marine bacterium is small. Aequorea The jellyfish Aeqzmrea aeqzcorea exhibits a bioluminescence that is not dependent on molecular oxygen. The bioluminescence arises from a conjugated protein (a photoprotein) of relative molecular mass 23000 daltons called aequorin. This interacts with calcium ions (and to a lesser extent with strontium ions) to produce light. Aequorin contains a prosthetic group coelenterazine which is converted into coelenteramide during the bioluminescent reaction. The aequorin coelenteramide complex is known as blue-fluorescent protein and it is the excited state of protein-bound coelenteramide which is the light emitter in this system.Aequorin + 2Ca2+ + blue-fluorescent protein 2Ca + CO + light (Amax. 470 nrn) Several workers have published detailed descriptions of the isolation and purification of aeq~orin.~' It is also available commercially. Renilla Anthazoan coelenterates such as the sea pansy Renilla reniforms exhibit a blue bio-luminescence due to an oxygen-dependent luciferin - luciferase type reaction. Renilla luciferase is a very hydrophobic glycoprotein (relative molecular mass 35 000 daltons) , whereas luciferin has the structure shown in Fig. 2. 3,5'-Diphosphoadenosine is required for bioluminescence as it is involved in the reversible conversion of luciferyl sulphate to luciferin by luciferin sulphokinase.Luciferin sulphokinase Luciferyl sulphate + PAP -- -+ luciferin + PAPS Luci f erase Luciferin + 0 ~ -+ oxyluciferin + CO + light (Amax. 480 nm) This enzyme can be isolated together with luciferase from Renilla. Yields of luciferyl sulphate from the organism are low and a synthetic alternative benzyl luciferyl sulphate, can be used to replace the natural luciferin. I n vivo the light emission from Renilla is green (Amax. 508 nm; cf. in vitro Amax. 480 nm) owing to the involvement of a protein (two identical sub-units of relative molecular mass 27 000 daltons) known as green-fluorescent protein. This is involved in an energy-transfer complex with luciferase and excited-state oxyluciferin bound to luciferase. Excitation energy is transferred from the excited-state oxyluciferin to a chromophore on the green-fluorescent protein which then emits green light November 1983 AND BIOLUMINESCENT METHODS.A REVIEW 1281 Further details of the mechanism of in vivo bioluminescence can be found in a paper by Cormier .38 Pholas This mollusc contains a luciferin - luciferase system which produces bioluminescence with an emission maximum around 490 nm. PhoZas luciferin is an acidic glycoprotein (relative molecular mass 34600 daltons) with an as yet unknown prosthetic group responsible for light emission. The luciferase is a dimeric glycoprotein consisting of two identical sub-units of relative molecular mass 150000 daltons. It contains two atoms of copper per molecule of enzyme and exhibits peroxidase activity.39 At one time this mollusc was widely distributed throughout the Mediterranean and along the Atlantic coast of Europe.Unfortunately it is now facing extinction mainly owing to coastal pollution. The impending loss of this species is of particular concern as the luciferin - luciferase system has considerable analytical potential e.g. micro-determination of peroxidase. Hopefully this species or the genes responsible for the production of its luciferase and luciferin proteins will be saved from extinc-tion thus allowing the study and exploitation of its biolumine~cence.~0 Instrumentation The basic elements of a luminometer (light-measuring instrument) are a light-tight detec-tion chamber and a light detector. Photomultiplier tubes and silicon photoelectrodes are the most popular types of detector although photographic film can also be used.41 Important considerations for photomultiplier tubes are the spectral response and sensi-tivity (signal to dark current ratio).The wavelength of maximum light emission depends on the particular luminescent reaction and on the experimental conditions (e.g. metal ions or acidic conditions shift the maximum emission from the firefly reaction from 560 to 615 nm42). Hence photomultiplier tubes must be carefully selected to have an adequate response a t the particular wavelength of light to be measured. Most analytical applications of luminescence simply require the measurement of either peak light intensity or the area under all or part of the light emission - time curve. A range of luminometers are available commercially; these include models that are battery-operated and therefore suitable for field work and some that are automatic or semi-automatic.Appropriately modified continuous flow ,43 flow inj and centri-fugal analysers46947 can also be used for luminescent assays. Remote sensing of luminescence in a reaction vessel by means of a fibre-optic probe has also been d o c ~ m e n t e d . ~ ~ For a detailed account of the various commercial luminometers the reader is directed to the recent review by Stanley.2 References to more sophisticated luminometers based on vidicon tubes and photodiode arrays are contained in reviews by Wehry.49,50 Reagents Chemiluminescent substances such as luminol isoluminol and lucigenin are readily obtainable from many chemical suppliers.In addition a range of isoluminol derivatives has become available from LKB Wallac Oy. The range of bioluminescent reagents commercially available is restricted to firefly luciferase luciferin (both synthetic and from firefly lanterns), bacterial luciferase NAD(P)H FMN oxidoreductase and aequorin. Leach4 has compiled a list of commercial sources of firefly luciferase and luciferin. Luminescent Assays Advantages and Disadvantages Assays based on a luminescent end-point combine the advantages of speed and sensitivity. In most luminescent assays the peak light emission occurs in less than 1 min and in some the peak is reached in less than 1 s (e.g. obelin lumine~cence).~~ Usually the peak light emission is proportional to analyte concentration but occasionally it is necessary to measure total light emission for several minutes or the kinetics of light emission.A luminescent assay for peroxidase illustrates the improvement in speed of analysis that may be obtained compared with conventional assays. Colorimetric assays for peroxidase based on either peroxide - 2,2’-azinodi (3-e t hylbenzt hiazoline-6-sulphonate) (ABTS) or peroxide - o-phenylene 1282 KRICKA AND THORPE CHEMILUMINESCENT Analyst Vol. 108 diamine (OPD) require incubation times with the oxidant and chromogen ranging from 15 min to 24 h but typically 30 min.52 Comparable and under some conditions superior sensitivity is achievable using a 30-s luminescent assay.40 The sensitivity of luminescent assays for nucleotides (ATP NADH NADPH) enzymes (peroxidase) and chemiluminescent substances (luminol acridinium compounds) is well documented For example ATP is easily detected at levels down to 500 fmol-1 pmol with conventional luminometers whereas acridinium compounds can be detected in attomole amounts using more sophisticated apparatus.53 An added advantage of luminescent assays is the wide range of linear response to analyte concentration; linearity is usually observed over several orders of magnitude.Most reagents used in luminescent assays present no greater danger to health and safety than those currently employed in the laboratory. In some instances luminescent reagents offer a safer alternative to potentially hazardous reagents (e.g. radioisotopes; see Immuno-assay). The main disadvantages of analytical methods based on luminescent end-points are interferences and at present the availability of some reagents.Practical Considerations and reagent purity. light levels and/or irreproducible results. The extreme sensitivity of luminescent techniques imposes stringent criteria of cleanliness Failure to monitor and control these factors results in high background Water Certain metal ions act as catalysts for chemiluminescent reactions (e.g. Co2+ and luminol-peroxide) whilst others cause a shift in wavelength of maximum light emission (e.g. Zn2+ and the firefly luciferase reaction). Hence water used to prepare buffers and other solutions used in luminescent assays should be free from metal ions. These are usually removed by distil-lation followed by passage down an ion-exchange column.This produces water of acceptable quality for most chemiluminescent or bioluminescent assays. Buflers A wide range of buffers is available for controlling the hydrogen-ion concentration during a chemical reaction. Individual buffers can exert a profound influence on the light emission from both chemiluminescent and bioluminescent reactions. This is illustrated in Table II(A) , which shows the relative light emission for an NADH assay using bacterial luciferase and NADH FMN oxidoreductase co-immobilised on to Sepharose carried out in different buffers. Both inter- and intra-buff er variations in light emission occur. The variability is presumably due to variations in purity (presence of metal ions) and interference by the buffer ions in the TABLE I1 EFFECT OF DIFFERENT BUFFERS ON LIGRT EMISSION FROM (A) A BIOLUMINESCENT AND (B) A CHEMILUMINESCENT REACTION (RELATIVE LIGHT EMISSION yo) Taken from Kricka L.J. Wienhausen G. K. and DeLuca M. unpublished data; and Stott R. A. unpublished data. Buffers* Assay ‘Tris TES HEPES Glycylglycine Phosphate’ (A) NADH assay using co-immobilised bacterial luciferase and NADH FMN oxido-reductase . . . . 72 34 31 12 100 Tris Tricine Glycine Borate AMPD (B) Assay for 8-microperoxidase using luminol and perborate . . 76 55 31 76 100 * TES = N-tris(hydroxymethy1) methyl-2-methaneaminopropanesulphonic acid ; Tris = tris(hydroxy-methy1)aminomethane; HEPES N-2-hydroxyethylpiperazine-N’-2-ethanesulphonic acid; Tricine = N-tris(hydroxymethy1)methylglycine ; AMPD = 2-amino-2-methylpropane-1,3-diol November 1983 AND BIOLUMINESCENT METHODS.A REVIEW 1283 light-producing reactions. Similar buffer sensitivity has been observed in chemiluminescent reactions [Table II(B)] and these results underline the need to scrutinise carefully assay buffers (buffer-dependent effects of this type have also been observed in cell culture experi-ment~~*). Cofactors Bioluminescent assays often utilise cofactors such as NAD(P) NAD(P)H ATP or ADP. Impurities in these substances can limit the sensitivity of such assays. For example some reputable NAD preparations may contain up to 0.006% of NADH. In assays based on bioluminescent measurement of NADH generated from NAD the presence of NADH as a contaminant of NAD generates a background bioluminescence that restricts the sensitivity of the assay.Aaxiliary enzymes In luminescent assays based on coupled enzyme systems contamination of the auxiliary enzymes with trace activities of other enzymes renders the assay non-specific. For example, malate dehydrogenase and aldehyde dehydrogenase activities may be encountered in some “purified” enzyme preparations. Containers Calcium contained in glass can be troublesome in assays using a e q ~ o r i n ~ ~ as it slowly leaches from the glass and reduces the luminescent activity of this protein. Thus plastic containers are preferred for its handling and storage. Cuvettes or reaction tubes should not be exposed to fluorescent lighting as this may induce phosphorescence and lead to high background light levels2 Light detection devices It is important to ensure that the light-detecting device e.g.a photomultiplier tube is sensitive to the wavelength of light to be detected. Most photomultiplier tubes in common use show good sensitivity in the region 340-450 nm but are less efficient at longer wave-lengths. Mixing Very fast luminescent reactions require both rapid highly reproducible mixing in front of Irreproducible mixing can the detector and instrumentation with a short response time. lead to poor analytical precision.2 Applications These have been chosen to illustrate the diversity of applications of analytical methods based on luminescent end-points. This section presents a selection of luminescent assays. Gas-phase Luminescent Reactions range of 1 p.p.b.-10 p.p.m.is generally required.56 of 0.03 p.p.b. using the chemiluminescent reaction of nitrogen oxide with ozone.57 Most interest has centred around the monitoring of air pollutants and an analytical working Nitrogen oxide can be detected at levels Nitrates nitrites and nitrosamines can also be assayed using reduction to nitrogen oxide and subsequent reaction with o ~ o n e . ~ ~ - ~ ~ Such assays have good precision (24%) and excellent sensitivity (nitrate 5 p.p.b. nitrite 0.05 p.p.b.). The reactions of nickel carbonyl with ozone and carbon monoxide,61 sulphur dioxide with oxygen atoms56 and hydrocarbons (e.g. ethylene) with are accompanied by the emission of light and this can be used to quantitate the individual reactants 1284 KRICKA AND THORPE CHEMILUMINESCENT Analyst Vol.108 Ni(CO) + 0 -f NiO + products NiO + CO Ni + 0, NiO* 2C,H + 20 -+ 4HCHO + 0 + light (Amax. 435 nm) SO + 2 0 -+ Ni + CO, -+ NiO* + 0, -+ NiO + light (Amax 550-600 nm) -+ SO + 0 + light (Amax. 300 nm) Nickel carbonyl can for example be assayed at 2 p.p.b. levels.61 Numerous other chemi-luminescent analyses have been accomplished in the gas phase ; for more detailed information the reader is referred to previous review^.^^-^^ Luminescent Reactions in the Liquid Phase Assays for metals and this has been exploited in analysis.65 Chemiluminescent and bioluminescent reactions are sensitive to a variety of metal ions Calcium. Aequorin bioluminescence is dependent on the concentration of ionised calcium and as little as 0.1 pmo11-1 can be detected.The assay is relatively specific for calcium, although under some conditions silver and magnesium ions may interfere. One of its dis-advantages is that light emission is not linearly related to calcium-ion concentration. Most interest in the assay has been centred on its use for measuring calcium ions inside living cells, and this application has been reviewed by Hallet and Campbell.51 Magnesiwz. Assays for magnesium ions based on the dependence of the bioluminescent firefly luciferase reaction on this ion have not proved very successful. Concentrations of magnesium as low as 60 pmol 1-1 can be detected but the assay is prone to interferences e.g., from free acids and heparin and could not be used for serum or plasma magnesium measure-ments.66 Zinc.Zinc ions can be assayed via their ability to activate a zinc-dependent enzyme. In this novel procedure a preparation of immobilised pyruvate oxidase is deactivated by treat-ment with EDTA which removes enzyme-bound zinc ions. The deactivated enzyme is then exposed to the sample. Addition of substrate produces peroxide and the latter is quantitated by means of its chemiluminescent oxidation of TCPO (Fig. 3). The detection limit for this assay is 8 pg and it is reported to be relatively free from interference^.^' Zinc ions present in the sample reactivate the enzyme. Transition metals. Chemiluminescent oxidation reactions of luminol lophine and luci-genin and their analogues are enhanced by many of tlie transition metals and the main problem in using these reactions as the basis of assays for metal ions is to achieve a degree of specificity.This has been accomplished in a number of ways. For lophine chemi-luminescence reaction conditions can be chosen that maximise chemiluminescent enhance-ment owing to a particular metal ion and under such conditions it is possible to detect 8 x 10-7moll-1 of Co(II) 5 x 10-6niol1-1 of Cr(II1) and 5 x 10-6mo11-1 of Cu(II).12 An alternative method of introducing specificity is to use a complexing agent. Chromium(II1) can be determined in complex mixtures by adding the chelating agent EDTA which com-plexes metal ions present in the sample and reduces their ability to enhance the chemi-luminescent oxidation of luminol. However Cr(V1) - EDTA complexes are formed relatively slowly at pH 4.4 and ambient temperatures so initially the chromium ions retain their ability to enhance luminol chemiluminescence.68 Specificity can also be introduced into chemiluminescent assays for metal ions by structural modification of the chemiluininescent compound.For example the 5-hydroxy analogue of luminol is reported to be superior to luminol for the detection of Fe(II1) and Co(I1). Detection limits for these nietal ions were 0.3 ng ml-l and 0.6 pg ml-1 respectively and in a limited study the precision (coefficient of variation) was 15%. High concentrations relative t o Co(II) of Zn Pb Al bIg Cr Ni Fe or Jln ions did not interfere in the a.w?!r:69 Dilution of samples as a way to minimise interferences is limited by tlie concentration o November l!H3 AND BIOLUMINESCENT METHODS.A REVIEW mruvate EDTA & mruvate oxidase - Zn2+ oxidase ‘ A I 1285 Pvruvic acid /- ’4 J phosphate Acetyl phosphate + H202 Light (Light a [Zn2’l) Fig. 3. Chemiluminescent assay for zinc. the metal ion and its luminescent detection limit. Interference due to Mg(I1) in an assay for Cr(II1) in seawater can be reduced by dilution of samples. Bromide is also added to enhance the luminescent signal from the Cr(II1) - luminol- peroxide reaction. In the presence of 0.5 M Br- the detection limit for Cr(II1) is 3.3 x loA9 M.70 Pre-concentration or “clean-up” of specimens by a chromatographic procedure is a more versatile method for removing interfering cations. Ion-exchange chromatography (Chelex-100 resin) has been used to separate Co(I1) from interfering Ca(II) Mg(II) Fe(III) Mn(I1) and Al(II1) ions in a luminescent assay based on the Co(I1)-enhanced luminescent reaction of lophine with per~xide.~’ The chromatographic step could be accomplished in 20 min with 99.5% recovery of Co(II) and the results obtained for the determination of Co(I1) in a National Bureau of Standards bovine liver specimen compared favourably with those obtained by neutron activation analysis.A similar procedure has been described for Cr(V1) analysis which used a Bio-Rex-5 ion-exchange column for pre-concentration and/or clean-up of samples. The detection limit for Cr(V1) in this luminescent method was 0.015 pg l-1.72 Enzyme assays About 10% of the 2000 enzymes known to date are ADP- or ATP-dependent and a further 10% are NAI>- or NADP-dependent.Hence the activity of these enzymes may be measured bioluminescently by means of either firefly luciferase or the bacterial luciferase - oxido-reductase system. Table I11 presents a list of the reported bioluminescent enzyme assays. A bioluminescent assay for creatine kinase (E.C. 2.7.3.2) illustrates the general principle of this kind of coupled assay (reagents for determining creatine kinase activity in serum are available in kit form from LKB Wallac Oy). Creatine kinase catalyses the reaction of ADP and creatine phosphate to produce ATP and creatine. Creatine kinase Creatine phosphate + ADP - -+ creatine + ATP Firefly luciferase luciferin Mg2+ ATP -f ligh 1286 KRICKA AND THORPE CHEMILUMINESCENT Analyst Vol. 108 If ADP and creatine phosphate are in excess then the amount of product formed is pro-portional to the creatine kinase activity.One of the products ATP is a cofactor for the firefly luciferase reaction and in the presence of an excess of firefly luciferase and luciferin it reacts to produce light. The intensity of the light is proportional to the ATP concentration, which is in turn proportional to the creatine kinase activity. If serum creatine kinase activity is to be measured it is first necessary to eliminate interference from other ADP-dependent enzymes particularly adenylate kinase (E.C. 2.7.4.3). This is accomplished using P,P5-di(adenosine-5')-pentaphosphate which is a potent inhibitor of this enzyme. The bioluminescent assay for creatine kinase correlates well with a spectrophotometric assay for the enzyme and the within-batch precision ranged in one study from 3 to 14y077 and in another study from 6 to 9%.78 Measurement of creatine kinase activity is used as a screening test for the detection of neonatal Duchenne muscular dystrophy and recently the bio-luminescent creatine kinase assay has been successfully applied to whole-blood specimens spotted on to filter-paper TABLE I11 LUMINESCENT ENZYME ASSAYS E.C.Enzyme number Reference Alcohol dehydrogenase CAMP phosphodiesterase ATP-phosphori bos yl transf erase ATP sulphurylase . . Creatine kinase . . Glucose oxidase . . Glucose-6-phosphate dehydrogenase . . Glycerol kinase . . Hexokinase . . . . 1.1.1.1 . . 3.1.4.c . . 2.4.2.17 . . 2.7.7.4 . . 2.7.3.2 . .1.1.3.4 . . 1.1.1.49 . . 2.7.1.30 . . 2.7.1.1 73 74 75 76 77-81 40 73 82 73 E.C. Enzyme number Reference D-S-H ydroxybut yrate dehydrogenase . . Isocitrate dehydrogenase Lactate dehydrogenase Malate dehydrogenase Peroxidase . . Pyruvate kinase . . Superoxide dismutase Lipase . . Myokinase . . . . Trypsin . . . . 1.1.1.30 . . 1.1.1.42 . . 1.1.1.28 . . 3.1.1.3 . . 1.1.1.37 . . 2.7.4.3 . . 1.11.1.7 . . 2.7.1.40 . . 1.15.1.1 . . 3.4.21.4 83 84 73 85 73 86 87 88 89 90 91 Similarly NAD(P)-dependent enzymes can be assayed using an excess of bacterial luci-ferase oxidoreductase FMN and a long-chain aldehyde. A sensitive assay for D-3-hydroxy-butyrate dehydrogenase (E.C. 1.1.1.30) in tissue specimens illustrates the usefulness of this type of bioluminescent assay.83 A sample of tissue (0.1-0.2 pg) isolated by hand dissection from a 20-pm thick section is incubated (0.5 h 37 "C) with D-3-hydroxybutyrate and NADf in the appropriate buffer (total volume 10.4 pl).Enzyme activity is then destroyed by heat inactivation and the NADH formed is detected by the addition of a mixture of bacterial luciferase oxidoreductase FMN and tetradecanal. This method is very sensitive (less than 0.2 pmol of NADH formed in the incubation of the enzyme with its substrate can be detected) and it is not subject to the non-specific background problems that plague conventional fluorimetric assays for NADH. Substances with peroxidase activity such as horseradish peroxidase (E.C. 1 .11.1.7) micro-peroxidase or haemoglobin can be assayed directly using an excess of luminol and an oxidant (peroxide perborate) under a variety of conditions (pH 6.5->12).At high pH (>12) it is possible to measure both active and inactive enzyme as under these conditions catalysis of the luminol reaction depends on the haem content of the protein rather than its enzyme activity.92 The detection limit for microperoxidase using the high-pH luminol- perborate reaction is 1.7 fmol (unpublished observations). The application of luminescent peroxidase assays is considered in the Immunoassay section. Oxidase enzymes (e.g. xanthine oxidase, glucose oxidase) can also be assayed by monitoring the production of hydrogen peroxide using the luminol TCPO or lucigenin reacti~n.~O An unusual type of enzyme assay has been developed for proteases in which a bioluminescent enzyme is used as a protease substrate.Incubation of a sample containing protease activity with bacterial luciferase results in proteolysis of the luciferase and loss of its enzymatic activity. Measurement of luciferase activity with FMNH before and after exposure to the sample gives a measure of its protease activity.9 November 1983 AND BIOLUMINESCENT METHODS. A REVIEW 1287 Assays for carbohydrates Reducing agents produce chemiluminescence when mixed with an alkaline solution of lucigenin. This reaction has been applied to a direct assay for reducing sugars and the following detection limits have been reported ascorbic acid 0.17 glucuronic acid 9.1 lactose 21 and glucose 21 mg 1-1.Metal ions particularly Cu(I1) and Fe(III) interfere and this limits the application of this assay for carbohydrates in complex specimens (e.g. serum, urine) .94 Carbohydrates can also be assayed luminescently by making use of specific enzyme re-actions. Glucose can be assayed via the chemiluminescent detection [luminol- hexacyano-ferrate(III)] of peroxide produced by the action of glucose oxidase (E.C. 1.1.3.4) on glucose, or via the bioluminescent detection (bacterial luciferase - oxidoreductase) of NADH formed in the reaction of glucose with hexokinase (E.C. 2.7.1.1.) and the subsequent reaction of the product glucose-6-phosphate with glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49). A detailed study of the former method for urinary glucose determinations found it to be accurate (average recovery of glucose added to glucose free urine = 100.5 & 3.373 and to correlate well with conventional colorimetric procedures (correlation coefficient 0.96).Interference due to uric acid (it reduces the peroxide formed in the enzymatic oxidation of glucose) was removed by treating urine with a mixture of barium hydroxide and zinc s ~ l p h a t e . ~ ~ Assays for nucleotides Table IV lists the different nucleotides for which bioluminescent assays have been devised. NADH and FMN can be assayed rapidly and with great sensitivity (femtomole-picomole amounts) using NADH FMN oxidoreductase coupled to bacterial luciferase. An NADPH-dependent oxidoreductase is also known and this can be used for an NADPH assay.98 NADH derivatives e.g.reduced nicotinamide-6-(2-aminoethylamine)purine dinucleotide with a 2,4-dinitrophenyl or biotinyl substituent on the 2-amino group can also be assayed (see Table VI).lo3 The use of firefly luciferase and luciferin as reagents for an ATP assay has already been mentioned. The firefly reaction can also be used as an indicator reaction for complex coupled enzyme reactions that allow the quantitation of purine and pyrimidine nucleotides. GMP GDP and GTP can be assayed in this manner and a detailed experimental procedure has been published.96 It should be noted however that in some instances the assay relies on other enzyme activities (e.g. transphosphorylating enzyme) contained in crude firefly luciferase - luciferin preparations The reaction of benzyl luciferyl sulphate with PAP and the subsequent bioluminescent reaction of the product luciferin with Renilla luciferase (see under Renilla) provides a very sensitive assay for PAP.As little as 100 fmol of PAP can be detected which represents a 50-fold improvement over conventional colorimetric techniques for assaying this c0mpound.1~~ TABLE IV LUMINESCENT ASSAYS FOR NUCLEOTIDES Nucleotide Reference AMP . . . . 96 CAMP . . . . . 97 ADP . . . . . . . . 86 ATP . . . . . . 42 CTP . . . . . . . . 86 FMN . . . . . . . . 98 FMNH . . . . . . 99 GMP . . . . . . 96 cGMP . . 96 Nucleotide GDP . . . . . . GTP . . . . . . NAD . . . . . . NADH . . . . NADP . . . . NADPH . . PAP . . . . PAPS . . . . UTP Reference 96 96 100 98 101 98 102 102 86 Steroid assays Luminescent assays for steroids can be accomplished using a specific enzyme or via an immunoassay using specific antibodies and a steroid labelled with a chemiluminescent ccm-pound.The use of steroid dehydrogenases is dealt with under Immobilised Luminescent Reagents. A steroid oxidase may also be used and some details of a luminescent cholesterol assay have been published by Burtis et al.lo5 Cholesterol oxidase oxidises cholesterol to cholestanone with a stoicheiometric production of peroxide. The peroxide was detecte 1288 KRICKA AND THORPE CHEMILUMINESCENT Analyst Vol 108 luminescently using a luminol - hexacyanoferrate( 111) reaction and a linear relationship between the integrated light emission and cholesterol was obtained at levels up to 250 g 1-1 (6.46 mmol l-l).Assays for drugs An example of a luminescent immunoassay for the cytotoxic methotrexate is cited under Immunoassay. Antibiotics such as chloramphenicol can be determined using the specific enzyme chloramphenicol acetyl transferase (E.C. 2.3.1.99) and coupling this reaction via acetyl-CoA synthetase (E.C. 6.2.1.1 .) to bioluminescent ATP quantitation.lo6 An alternative approach to drug assay is to use drug-sensitive organisms. A test sample is incubated with a broth culture of the particular drug-sensitive bacterium. The presence of the drug suppresses bacterial growth (in proportion to drug concentration) and this is reflected in the amount of ATP extractable from the culture. The extracted ATP is quantitated using the bioluminescent firefly reaction.A range of antibiotics have been detected in this manner, e.g. gentamicin using Klebsiella edwardsii var. atlantae cephaloridine using Staphylococczls aureus and rifampicin using Sarcina lutea as the test organism. The detection limit of the assay for rifampicin in serum was 25 ng ml-l.lo7 Immobilised Luminescent Reagents A variety of methods are available for immobilising molecules and they can be broadly classified into methods based on physical and on chemical processes.lo8 In the former, molecules of the reagent are adsorbed to the surface or entrapped within an insoluble solid support or matrix. Chemical methods involve the formation of covalent bonds between molecules of the reagent and reactive groups on a solid support.Both types of immobilisa-tion method have been used to prepare immobilised luminescent reagents. Specific advan-tages associated with immobilised reagents are re-usability improved stability and increased efficiency. Chemiluminescent reagents Paper impregnated with luminol (0.1%) and a catalyst copper(I1) sulphate (0.01%) has been used as a solid-phase indicator for hydrogen peroxide in gas mixtures at concentrations of 0.1-10pgm-3. The assay is not subject to interference by other gases such as ozone, carbon monoxide or sulphur dioxide or organic substances (e.g. benzene acetone) .log Luminol-impregnated membranes coupled with peroxidase - glucose oxidase-impregnated paper pads and an instant photographic detection system have been applied to the analysis of glucose.41 Because of the exposure latitude of the Polaroid film used for light detection, the assay provided a “threshold”-type test-an amount of glucose above 28 nmol exposed the photographic emulsion fully whereas below this amount the emulsion was unexposed.A novel application of chemically immobilised isoluminol has been in an assay for serum cholinesterase (E.C. 3.1.1 .S) .l10 Normally this enzyme is assayed by colorimetric determina-tion of a thiol produced by enzymatic cleavage of an acetylcholine thioester. Alternatively, this thiol can react with isoluminol immobilised on to Sepharose via a disulphide linkage. A thiol - disulphide interchange releases soluble isoluniinol and this is assayed luminescently. The assay requires a 10-pl sample of specimen has a detection limit in the nanomole-pico-mole range and is not influenced by other components of serum.Luminol and 9-carboxy-m-chlorophenolate acridane derivatives of Sepharose have also been prepared and used in thiol - disulphide exchange reactions for the assay of piconiole amounts of thiols.lll Very sensitive assays for proteolytic enzymes are possible using immobilised luminogenic substrates.l12 The analytical principle is shown in Fig. 4. Proteolytic action of the enzyme releases soluble isoluminol from the immobilised luminogenic substrates and this is then assayed using peroxide and haematin at high pH. Assays for a-cliymotrypsin (E.C. 3.4.21.1.) , trypsin (E.C. 3.4.21.4) and thrombin have been devised and these have detection limits of 0.1, 0.01 and 0.5 ng respectively.Analytically they are also more convenient than their soluble counterparts. Bioluminescent reageizts Chemical ininiobilisation of bioluminescent enzymes such as firefly luciferase and bacterial luciferase - NAD(P)H FBfN oxidoreductase to glass beads or r 0 d ~ ~ ~ y ~ 1 ~ Sepharose particles11 November 1983 AND BIOLUMINESCENT METHODS. A REVIEW 1289 Affi-gel 10 - peptide - isoluminol Proteinase 1 Affi-gel 10 - peptide-C02H + isoluminol (i) Separate released isoluminol (ii) Haematin - H2O2 (pH > 11) 1 Light oc proteinase activity Fig. 4. Cheniiluminescent assay for proteinase. and cellophane films115 has produced active immobilised enzymes. The most active prepara-tions have been those produced using cyanogen bromide-activated Sepharose 4B or CL6B as the solid support.Picomole-femtomole amounts of ATP or NAD(P)H can be detected using immobilised firefly luciferase and bacterial luciferase - oxidoreductase respectively. The range of assays possible with this type of reagent can be extended by co-immobilising other enzymes along with the bioluminescent enzymes. For example 3a,3/3-steroid dehydro-genase can be co-immobilised with bacterial luciferase and NADH FMN oxidoreductase on to Sepharose and the coupled enzyme system used to assay androsterone or testosterone in picomolar amounts (Fig. 5) .l14 Other substrates of dehydrogenase enzymes can be assayed in a similar fashion and these are summarised in Table V. The most extensively tested of these immobilised bioluminescent enzyme systems has been co-immobilised 7a-hydroxy-steroid dehydrogenase - bacterial luciferase - NADH FMN oxidoreductase.118 This is the principal reagent in an assay for serum primary bile acids (cholate chenodeoxycholate and their glycine and taurine conjugates).The assay has a detection limit of 0.5 pmol shows good reproducibility and compares well with the much slower conventional assays. Proteins interfere in the assay but this can be overcome by diluting samples. The co-immobilised enzyme preparation is stable (no detectable loss in activity when stored in a glycerol - buffer -dithiothreitol mix at -20 "C) and can be re-used. This latter feature of immobilised enzymes has been exploited in an automated system for bile acid analysis based on a continuous flow system and small flow cells (200-500 p1 volume) packed with co-immobilised 7a-hydroxy-steroid dehydrogenase - bacterial luciferase - NADH FMN oxidoredu~tase.~~~ In contrast to the above attempts to immobilise bioluminescent enzymes by physical methods have been less successful.The most notable success in this area has been the use of intact bioluminescent marine bacteria (Photobacterium Jischeri strain M J-1) confined by a semipermeable polypropylene or PTFE membrane as an oxygen sensor. The bioluminescence Bacterial luciferase NADH - FMN oxidoreductase 3a-Hydroxysteroid dehydrogenase Light Deca na I i FMNH2 kN NADH t 5a- androstane--3,17-dione ' Androsterone t NAD Fig. 5. Bioluminescent assay for androsterone 1290 KRICKA AND THORPE CHEMILUMINESCENT Analyst Vol.108 of marine bacteria is oxygen dependent and this is the underlying principle of this sensor, The sensor functions for oxygen in the gas or liquid phase detects oxygen at concentrations above 0.04 pmol l-l shows good precision (coefficient of variation = 1.67% at 0.7 pmol l-1 of oxygen) and is reasonably stable (10% decline in response after 8 h at 20 OC).12* TABLE V ANALYTICAL APPLICATIONS OF IMMOBILISED BIOLUMINESCENT ENZYMES Immobilised enzymes Firefly luciferase . . . . . . . . . . . . Bacterial luciferase . . . . NAD(P)H FMN oxidoreductase bacterial luciferase . . Alcohol dehydrogenase NADH FMN oxidoreductase, bacterial luciferase . . . . Glucose-6-phosphate dehydrogenase NADH FMN oxidoreductase bacterial luciferase . . Alanine dehydrogenase NADH FMN oxidorkductase, bacterial luciferase .. . . . . . . . . Glutamate dehydrogenase NADH FMN oxidoreductase, bacterial luciferase . . . . . . . . . . . . Malate dehydrogenase NADH FMN oxidoreductase, bacterial luciferase . . . . . . . . Lactate dehydrogenase NADH FMN oxidoreductase, bacterial luciferase . . . . 6-Phosphogluconate dehydrogenase NADPH':'FMN oxidoreductase bacterial luciferase . . . . . . . . Hexokinase glucose-6-phosphate dehydrogenase, NADPH FMN oxidoreductase bacterial luciferase . . . . Analyte ATP FMNH, NAD(P)H Ethanol Glucose-6-phosphate L- Alanine L-Glu tamate L-Malate L-Lactate NAD 6-Phosphogluconate NADP D-Glucose Reference 116 116 99 73 73 117 117 117 117 117 117 Immunoassay The considerable sensitivity of luminescent reactions has led to their exploitation in different types of imrnunoassay.l2l Radioactive substances are usually used as the label in such assays but concern over the potential health hazards associated with radioactivity has stimulated the search for alternative and safer labels.Luminescent labels are just one of many alternatives that have been developed.122 Their advantages are that they can be quantitated rapidly are relatively stable have high specific activity and can take part in amplification reactions. The label can be a chemiluminescent substance such as luminol or a bioluminescent substance such as the enzyme luciferase. Particular problems are that there is a considerable loss of chemiluminescent efficiency and in enzyme activity respectively on linking to other molecules such as antigens or antibodies.Examples are peroxidase which is a catalyst of the chemiluminescent luminol- peroxide reaction and NAD which is a cofactor for the bioluminescent reaction catalysed by bacterial luciferase -oxidoreductase. An example of this application of luminescence in immunoassay is the use of dehydrogenase enzyme labels that produce NADH. This product is then monitored using the bioluminescent bacterial luciferase - oxidoreductase reaction. The most successful luminescent immunoassays have been those employing luminol iso-luminol or acridinium compounds as labels and assays based on the luminescent monitoring of peroxidase glucose-6-phosphate dehydrogenase and pyruvate kinase labels.Novel chemiluminescent labels such as 1,2-dioxetanes that simply require thermal activation have been prepared but no applications have been described.123 Table VI presents a selected list of luminescent immunoassays. An interesting feature of luminol and isoluminol labels is that the luminescence can be influenced by antigen antibody binding and this makes possible the development of homo-geneous or non-separation immunoassays. Binding can produce a change in the kinetics an enhancement or an inhibition of light emission. Thus it is possible to determine directly the extent of binding of a labelled antigen with an antibody without the need for separation. This type of assay is therefore much quicker to perform than conventional heterogeneous They can be employed in immunoassays in three different ways.1. 2. The label can be a catalyst or a cofactor for a luminescent reaction. 3. Products from labels can be monitored using a luminescent reaction November 1983 AND BIOLUMINESCENT METHODS. A REVIEW 1291 TABLE VI LUMINESCENT IMMUNOASSAYS Analyte Rabbit IgG Cortisol . . . . Thyroxine . . Human IgG . . 17P-Oestradiol . . l7g-Oestradiol . . Methotrexate Hepatitis B surface antigen . . Type of label or Luminol Chemiluminescent . . N-(4-Aminobuty1)- and N-ethylisoluminol bioluminescent . . N-(4-Aminobutyl)- labels N-ethylisoluminol . . N-(4-aminobutyl)-N-ethylisoluminol . . N-(4-Aminobutyl)-N-ethylisoluminol . . Acridinium-9-carboxyl chloride . . Firefly luciferase .. Fluorescein* Label assay Assay format Substance labelled Heterogeneous Antibody (IgG) Heterogeneous Cortisol Heterogeneous Thyroxine Heterogeneous Antibody (IgG) Heterogeneous 178-Oestradiol Heterogeneous 17F-Oestradiol Heterogeneous Methotrexate Heterogeneous Antibody (IgG) Dehydroepiandro-Albumin . . . . . . Horseradish Heterogeneous Antibody (IgG) Herpes simplex virus . . Horseradish Heterogeneous Antibody (IgG) sterone . . . . Horseradish Enzyme labels Heterogeneous Dehydroepiandro-peroxidase sterone peroxidase peroxidase Transferrin . . . . Pyruvate kinase Insulin . . . . Pyruvate kinase Human chorionic' somatomammotropin Glucose oxidase Heterogeneous Transferrin Heterogeneous Insulin Heterogeneous Antibody (IgG) Biotin . . . .. . Isoluniinol Homogeneous Homogeneous Biotin Progesterone . . . . N-(4-Aminobutyl)- assays Homogeneous Progesterone-ll-Phenytoin . . . . Glucose-6-phosphate Homogeneous Phenytoin N-e thylisoluminol hemisuccinate deh ydrogenase * Fluorescein determined via a chemiluminescent reaction with sodium hypochlorite. t PFU = plaque-forming unit. Sensitivity <5 ng ml-1 54 pg per tube 6.1 nmol I-' <1 ng per bead 1.5 pg per tube 50 fmol 0.5 pmol 0.1 ng ml-l 25 Pg 5 ng 40 PFUt per sample <0.1 ng per tube <5 mU 1-l 0.2 ng per tube 50 nmol l-l 5 pg per tube 30 ng Reference 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 (separation) immunoassays although the sensitivity is inferior and the assay prone to inter-ferences.Most of the luminescent immunoassays that have been described are for analytes of clinical interest. However antibodies can be raised to many different low and high relative mole-cular mass substances so that these types of luminescent immunoassays could be applied to a much wider range of compounds. An immunoassay described by Wannlund et aZ.141 for trinitrotoluene illustrates this point. It also has one of the lowest detection limits of any of the different luminescent immunoassays so far described-the assay can detect attomolar amounts of TNT. Analytical Applications of Intact Bioluminescent Organisms The exploitation of the oxygen dependence of bioluminescent marine bacteria in an oxygen sensor has already been mentioned (see Immobilised Luminescent Reagents).Intact marine bacteria can also be used in a novel screening test for mutagenic substances and substances that damage DNA.lg2 The test uses a dark variant of the marine bacterium Photobacterium Zeiognathi. On incubation with mutagens or DNA-damaging substances the in vivo bioluminescence of the bacteria is restored by mechanisms as yet not fully understood. The test is very sensitive ; for example the mutagen N-methyl-N-nitro-N-nitrosoguanidine can be detected at 0.002 pg ml-l. This is 50-100 times as sensitive as conventional tests, e.g. the Ames test is simple to perform and can be automated. Dim mutants of Beneckea lzarveyi and Photobacterium fzscheri exist that are stimulated to emit light on addition of certain aliphatic aldehydes and fatty acids.This forms the basis of a highly sensitive bioassay for these types of substance in picomolar a m 0 ~ n t s . l ~ ~ It simply involves mixing an aliquot of a cell culture with the sample and measuring the peak light emission which usually occurs in less than 1 min. The bioassay is not specific for example the bioluminescence of the dim mutant B. harveyi M17 is stimulated by tetra-decanoic acid and also by tridecanoic and pentadecanoic acids (relative luminescence intensity 100 56 45 respectively). Similar simple and rapid assays are available for phospholipases A and C.lg 1292 KRICKA AND THORPE CHEMILUMINESCENT Analyst Vol. 108 Luminous bacteria have also been employed as nuclear radiation and air pollution detec-t o r ~ ~ ~ ~ and in studying the actions of anaesthetics such as ha10thane.l~~ Luminescent Detectors in Chromatography Chemiluminescent and bioluminescent reactions have been employed in detection systems for several chromatographic techniques.Chemiluminescent reactions can for example be used to detect and quantitate ascorbic acid and other reducing compounds resolved by high-performance liquid chromatography (HPLC)147 and the selective determination of iodinated hydrocarbons resolved by gas chromatography (GC) .148 The bioluminescent firefly luciferase reaction has been used as a specific detector for creatine kinase isoenzymes separated by HPLC on a SynChropak AX300 column; however although the method is rapid and very sensitive, it does not have general applicability in chr~matography.~~~ Detection methods based on chemiluminescent reactions such as those between ozone and nitrogen oxide the reaction of hydrocarbons with “active nitrogen” and the reactions of fluorophors with TCPO - hydrogen peroxide have wider applicability.Lafleur and co-w o r k e r ~ ~ ~ ~ ~ ~ ~ ~ have described a pyrolysis - cliemiluminescent detector for nitroaromatic compounds separated by GC. The separated nitro compounds were pyrolysed at 800-900 “C and the nitrogen oxide formed detected via a cliemiluminescent reaction with ozone. The method showed good reproducibility and sensitivity “g. nanogram amounts of the explosives RDS (hexali ydro- 1,3,5-trini t ro-s- t riazine) and MNX (oct ah ydro- 1,3,5,7- tetra-nitro-1,3,5,7-tetrazocine) could be detected (coefficients of variation in the range 0.7-5.6%).Similar methods have also been described for the determination of atmospheric ammonia and trimethylamine using GC.152 A chemiluminescent aerosol spray detector has been developed based on ozone-induced chemiluminescence from a range of organic compounds.153 Compounds determined include azides (e,g. methyl red detection limit 0.42 ng) hydrazines (1 ,I-dimethylhydrazine 65 ng) nitrogen heterocycles (indole 39 ng) sulphur compounds (thiourea 880 ng) olefins (tra~zs-l,2-dichloroethylene 1 10 ng) and fluorescent substances (fluorescein 1.7 ng). In all a total of 60 organic compounds were tested and detection limits determined. Hydrocarbons separated by capillary column GC can be detected and quantitated using “active nitrogen,” which reacts to form excited-state CN molecules.Microgram amounts of propylene hexane and hept-1-ene and nanogram amounts of methane and vinyl fluoride have been measured in this In liquid chromatography poly-cyclic aromatic hydrocarbons have been detected using peroxyoxalate chemiluminescence.155 An HPLC detector capable of quantitating oxygen-containing compounds has been described in which the analytes such as aliphatic alcohols aldehydes ethers and saccharides are oxidised in a photochenlical reaction to produce hydrogen peroxide which is then quanti-tated using the cobalt (11) - luniinol chemiluminescence reaction. The detection limits for most analytes are in the microgram range.156 Derivatisation using fluorescent compounds is an established method for improving the detection of the resolved components of a mixture.The sensitivity of the detection can be improved 20-fold by using the cliemiluminescent reaction between TCPO peroxide and the fluorescent derivative rather than its direct fluorimetric detect ion. 157 9 lj8 This detect ion system has been employed in conjunction with HPLC for the measurement of dansylated amino acids in tlie 50 fmol-40 pmol range,157 and femtoniolar amounts of fluorescamine-labelled norepinephrine and dopamine.158 Localisation of luciferases and enzymes that produce substrates for luciferases has also been achieved in polyacrylamide gels by auto-l~mograpliy.1~9 Following electrophoresis or isoelectric focusing the gels were laid on photo-graphic film and luminescent reactions initiated with the appropriate co-reactants.Miscellaneous Reactions and Assays Luminescence has tlie potential to replace many of the analytical techniques in use today. Some broad areas of application not covered in this account include biomass determina-tion,160 the study of cellular events (q. pliagocytosis) ,161j162 assays based on inhibition, catalysis and quenching of luminescent reactions (e.g. assays for aldehydes,163 liaem proteins,164 mannitol and ~ o r b i t o l l ~ ~ ) assays for oxidants (e.g. chlorine,166 iodine,167 dissolved ~ x y g e n l ~ ~ - l ~ ~ ) titrimetry,65 material degradation (low levels of chemiluminescence occur whe November 1983 AND BIOLUMINESCENT METHODS. A REVIEW 1293 certain materials e.g. foods or organic polymers degradel’l) assays for anions (e.g.sulphide,21 hypochlorite21) amine~,l’~ and chemiluminescent reactions in flames.173 Conclusions Although known since the 1800s chemiluminescent and bioluminescent reactions are not in widespread routine use in the analytical laboratory. In the past this has been blamed on the lack of suitable equipment and either the unavailability or the poor quality of reagents. 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