Chemiluminescence in Analytical Chemistry

26	Garcı´a-Campan˜a et al.
Table 1 Chemiluminescent Systems in Redox Titrations
Indicator	Species	Titrating reagent
Luminol	OCl , OBr	As2 O3
	As(III), Sb(III), S2 O32 , SO32 , S2 , CN , SCN	NaOBr
	As(III), Sb(III), S2 O32	NaOCl
	As(III), SO32	I2
Lucigenin	Fe(CN)64 , As(III), OCl , OBr , Cr(III)	H2 O2
	OCl , OBr	N2 H4• H2 SO4
Siloxene	I , Fe(II), Sn(II), Mo(III), Ti(III), As(III), Pb(II),	MnO4
	C2 O42 , H2 O2, I2(a) IO3 (a,b), Ag (c), VO3 (d)
	Fe(II), As(III), I , C2 O42 , H2 O2	Ce(IV)
	Fe(II), Tl(I)	Cr2 O72
	Fe(II)	VO3

aAfter reduction with Zn to iodide.

bAfter addition of an excess of iodide solution of known concentration.

cAgI is precipitated by adding an excess of iodide, which is then titrated in the presence of the precipitate.

dAfter adding Fe(II) in excess.

excess was back-titrated with a standard solution of lucigenin [154]. Luminol in the presence of H2 O2 and Co(II) as a catalyst was used for analysis of aluminum in colored samples by titrating 8-hydroxyquinoline [155]. The endpoint is detected by the decrease of CL emission due to hydroxyquinoline removing Co(II) from its association with luminol.

In complexometric titrations with EDTA, luminol and lucigenin have been employed as indicators based on the fact that complexing the ions of a catalytic heavy metal can cause changes in the emission from these reactions in alkaline solution and in the presence of oxidizing agents. In the case of luminol, if the metals are titrated with EDTA, the endpoint is observed by the disappearance of the light originating from luminol. On the contrary, using lucigenin the CL emission indicates the end of the process. Ca(II), Sr(II), and Ba(II) were determined by direct titration using luminol [115] and Cu(II) [115], Cd(II), Zn(II), and Ni(II) using luminol or lucigenin [156]. Employing the indirect method, Pb(II) and Hg(II) were determined by adding EDTA in excess and back-titration with Cu(II) solution [150].

4.3Application to Firefly Luciferase Reaction in the Analysis of ATP

Although already in 1947 McElroy discovered that adenosine triphosphate (ATP) and magnesium were essential for the BL reactions by which fireflies produce

Historical Evolution of Chemiluminescence

27

light [157], the reaction mechanisms and kinetics were only reported much later

[158].Because of the importance of ATP in these mechanisms, the BL system was used as standard procedure for an extremely sensitive and selective determination of the cited molecule. The detection limit for the firefly assay method is about 10 11 mol/L ATP, allowing the detection of single cells in certain cases

[159].Since ATP is present in all organisms with a fairly constant intercellular concentration, the ATP concentration is a useful index of total biomass and has been applied as such to monitor bacteria in food, biomass in wastewater, and sewage sludge [160]. The high sensitivity of this assay allows the use of the firefly assay as a tool for detection of life on other planets. Table 2 shows some applications of the analysis of ATP.

4.4 The Application of Chemiluminescence in Immunoassay

Since the development of radioimmunoassay (RIA), many assays that rely on the specificity of the antigen-antibody binding reaction have been developed because of their inherent sensitivity and specificity. A typical competitive binding

Table 2 Some Applications in the Analysis of ATP

Direct determinations

Applications	Field
Monitoring of fermentation rates	Process control in food, beverage, and drug
	industries
Detection of bacterial contamination	Quality control in food, beverage, drug, and
	cosmetic industries
Measuring biomass	Treatments of wastewaters, oceanography
	studies
Detecting the presence of life	Extraterrestrial investigations
Control of cell viability	Medical
Measuring infections caused by bacteria	Medical
Indirect determinations
Species analyzed	Importance
Creatinine phosphate (CP ADP →	Energy reservoir for muscle activity
creatinine ATP)
AMP	Mediator of hormone activity
Dissolved O2	Measurement of water quality
	Medical
Inorganic pyrophosphate	Starting material for several biological com-
	pounds

28	Garcı´a-Campan˜a et al.

assay for the determination of an antigen (Ag) can be represented by the following scheme, where unlabeled and labeled antigen (Ag*) compete for sites on the antibody (Ab):

Ag Ag* Ab → AgAb Ag*Ab

After equilibration, the amount of bound and free-labeled antigen can be measured, and a calibration curve can be used to determine the analyte. Radioactive labels have been extensively used because of the sensitivity of the measurement; however, they have several disadvantages, such as the waste disposal problem and the unstable nature of reagents. CL tags were therefore considered attractive alternatives due to their low (excellent) detectability, which was not fully provided by most fluorescent labels.

In 1976, Schroeder et al. developed a CL immunoassay as alternative to RIA in the analysis of very low levels of biological substances [161]. They used isoluminol derivatives as fluorescent labels and specific protein bindings were monitored allowing the assay of biotin and thyroxin. Campbell and McCapra’s groups also studied the potential of acridinium esters as labels [162], being successfully used to achieve detection limits in the femtomole ranges. The phenyl carboxylate ester group was attached to the amine group on the antigen through a N-succinimide funtional group on the ester [163].

The PO CL detection system was also applied in immunoassay to measure the CL of fluorescent labels, but solvent restrictions significantly affected the precision. Arakawa et al. [164], in 1982, developed a sensitive, selective CL enzyme immunoassay of 17 α-hydroprogesterone using the glucose oxidase and TCPO-8-anilinonaphthalene-1-sulfonic acid system. Grayeski and Seitz used PO CL of rhodamine B isothiocyanate-labeled analytes for batch analysis and also as an alternative to radioisotope immunoassays [165]. Chapter 18 discusses practical aspects of enzyme immunoassay, the mechanism of enhanced CL using acridan esters as chemoluminogenic reagents, and its synthesis and recent applications.

4.5Chemiluminescence in Flow Analysis and Immobilization Techniques

The flow-cell design was introduced by Stieg and Nieman [166] in 1978 for analytical uses of CL. Burguera and Townshend [167] used the CL emission produced by the oxidation of alkylamines by benzoyl peroxide to determine aliphatic secondary and tertiary amines in chloroform or acetone. They tested various coiled flow cells for monitoring the CL emission produced by the cobalt-catalyzed oxidation of luminol by hydrogen peroxide and the fluoresceinsensitized oxidation of sulfide by sodium hypochlorite [168]. Rule and Seitz [169] reported one of the first applications of flow injection analysis (FIA) in the CL detection of peroxide with luminol in the presence of a copper ion catalyst. They

Historical Evolution of Chemiluminescence

29

envisaged routine applications in clinical analysis via coupling reactions that generate hydrogen peroxide. Chapter 12 reports the basic principles of FIA, instrumentation, recent FIA versions, and an extensive overview of the last analytical applications, mainly in drug analysis.

Immobilization techniques based on chemical or physical processes were introduced from the seventies onward to prepare immobilized luminescent reagents with specific advantages such as reusability, improved stability, and increased efficiency. Examples of CL reagents are luminol using a solid-phase indicator for hydrogen peroxide in gas mixtures utilizing paper impregnated with luminol [170], luminol-impregnated membranes coupled with peroxidaseglucose oxidase–impregnated paper pads and the instant photographic detection for the analysis of glucose [171], or isoluminol immobilized on sepharose via a disulfide linkage for the analysis of serum cholinesterase [172]. This enzyme produces a thiol by enzymatic cleavage of an acetylcholine thioester; the thiol reacts with immobilized isoluminol and a thiol-disulfide interchange releases soluble isoluminol, which is determined chemiluminescently. Luminol and 9- carboxy-m-chlorophenolate acridane derivatives of sepharose have also been prepared and used in thiol-disulfide exchange reactions for the assay of picomole amounts of thiol [173].

Chemical immobilization procedures of bioluminescent enzymes such as firefly luciferase and bacterial luciferase-NAD(P)H:FMN oxidoreductase to glass beads or rods [174, 175], sepharose particles [176], and cellophane films [177] have produced active immobilized enzymes. Picomole-femtomole amounts of ATP or NAD(P)H could be detected using immobilized firefly luciferase or bacterial luciferase-oxidoreductase, respectively.

4.6First Uses of Chemiluminescence as a Detection Technique in HPLC

Promising perspectives introduced the first applications of CL reactions as detection mode in liquid chromatography (LC), combining the high efficiency in separation and the low detection limits inherent to several CL systems. The luminol CL system was applied as postcolumn detection mode in HPLC by Kawasaki et al. [178]. They used labeled amines and carboxylic acids with an isoluminol derivative prior to separation by HPLC; postcolumn reaction with hydrogen peroxide and potassium ferricyanide enables the application of CL detection. The postcolumn CL reaction of lucigenin with glucose [179], heparin [180], cortisol, and carboxylic acid p-nitrophenacyl esters [181] allowed sensitive detection of these analytes.

Outstanding research—among others—on the use of aryl oxalates for CL detection in high-performance liquid chromatography (HPLC) was introduced in the 1980s by Imai’s group from Tokyo University. They determined fluorescers

30	Garcı´a-Campan˜a et al.

or fluorophore-labeled compounds by HPLC and CL detection [182], using TCPO in ethyl acetate, and hydrogen peroxide in acetone, mixed with the column eluate using separate pumps and a suitable mixing vessel. Dansyl amino acids (alanine, glutamic acid, methionine, and norleucine) were separated on a -Bon- dapak C18 column and fmol limits could be reached. The experiments in static systems demonstrated that the CL intensity decays apparently according to a firstorder rate equation; so the measurements have to be carried out fast. Also, the authors state that care should be taken during CL detection in TLC experiments. In flow systems, on the other hand, reproducible peak heights that are proportional to the concentration of the fluorescers are obtained since the detector, which is a fluorescence spectrophotometer with the light source switched off, measures the CL intensity during a fixed stage of the reaction. An analogous TCPO–hydrogen peroxide system was used for determination of fluorescamine-labeled catecholamines in urine [183], providing a 25-fmol detection limit, which is about 20 times lower than that using conventional fluorescence detection. Several aryl oxalates were evaluated for the hydrogen peroxide–induced CL detection of various fluorescers for use in HPLC postcolumn reactors [184]. Different oxalate esters were synthesized for use in the PO-CL reaction and their characteristics were studied in relation to the introduction of alkoxy side chains in the phenyl ring and the solubility effects [185, 186].

The same group reported in 1986 a sensitive and selective HPLC method employing CL detection utilizing immobilized enzymes for simultaneous determination of acetylcholine and choline [187]. Both compounds were separated on a reversed-phase column, passed through an immobilized enzyme column (acetylcholine esterase and choline oxidase), and converted to hydrogen peroxide, which was subsequently detected by the PO-CL reaction. In this period, other advances in this area were carried out such as the combination of solid-state PO CL detection and postcolumn chemical reaction systems in LC [188] or the development of a new low-dispersion system for narrow-bore LC [189].

Features of CL detection in HPLC, CL reactions used, construction of devices, optimization of experimental conditions, and recent applications are discussed in Chapter 14.

4.7 Application of Chemiluminescence in DNA Analysis

In 1987, CL started to be applied in DNA hybridization assays as an alternative to the use of radioactive tags. These assays are based on the specificity of a binding process: that of DNA strands for each other. An unknown DNA can be identified with the Southern blot method in which the strands of the analyte are separated and allowed to interact with labeled probe DNA strands on nitrocellulose filter paper. If the label on the probe is detected, the DNA can be identified and, in some cases, quantitated. Conventionally, radioactive tags were used be-

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31

cause of the sensitivity required for quantitation. However, horseradish peroxidase (HRP) can be used as a DNA probe to be detected with the luminol CL reaction, using photographic detection. This alternative avoids the disadvantages from the use of radioactive material and the long time required by these processes; the endpoint is reached rapidly and levels as low as 1 pg of DNA can be easily detected. The CL assay was commercialized in 1985 [190].

In Chapter 19, basic aspects and recent trends in CL-based DNA analysis are discussed.

4.8 Chemiluminescence Sensors

In 1981 Freeman and Seitz reported on the first optical sensors to detect oxygen in the gas and liquid phases using the tetrakis(dimethylamino)ethylene oxidation reaction [191]. The reagent was held behind a Teflon poly(tetrafluoroethylene)- membrane through which oxygen diffused while light was transmitted to the photomultiplier detector. Detection limits and responses were similar to those of the oxygen electrode but the interferences from several species were removed because they do not affect the CL reaction. Aizawa et al. developed a sensor for analysis of hydrogen peroxide with HRP immobilized on a photodiode, which was immersed in an alkaline solution containing luminol and the analyte [192]. Also, glucose was determined with a bienzyme sensor containing both HRP and glucose oxidase.

Chapter 20 includes an extensive review of the CL sensors for determination of analytes in air, vapors, or liquids using different immobilization modes, its application to different analytes, based on enzyme or nonenzyme reactions, as well as CL immunosensors and DNA sensors.

4.9 Chemiluminescence Produced by Direct Oxidation

Until the end of the 1980s, CL analytical determinations involved interaction of the analyte with well-established CL reactions, often as a catalyst or as a sensitizer, or after a sequence of other reactions. However, Townshend’s group discovered new CL reactions by testing the analyte with a wide range of oxidants (and reductants) under a large range of reaction conditions [193]. The typical tested oxidants were H2 O2, ClO , Ce(IV), IO4 , MnO4 (in acid and alkaline medium), and Br2, allowing the possibility of adding catalysts and/or carrying out the reactions at different pH values. There are some guidelines for predicting which analytes are likely to generate CL. For example, if oxidation of the molecule is known to give a fluorescent product, or if the analyte itself has a structure that might lead to fluorescence behavior, there is a possibility that oxidation of the analyte will give CL. Some of the first applications were the CL determination of morphine [194], buprenorphine hydrochloride [195], and the benzodiazepine

32			Garcı´a-Campan˜a et al.
Table 3 CL Determinations Based on Direct Oxidation
Analyte	Oxidant system	Detection limit (M)		Ref.
Sulfide	ClO	1	10 7	197
	H2 O2-peroxidase	1.5 10 9		198
Adrenaline	H2 O2-OH	6	10 7	199
Humic acids	MnO4 -OH	0.7 mg/l		200
Paracetamol	Ce(IV)	4	10 7	201
Quinones	H2 O2-OH	1	10 5	202
Hydrazine	ClO	ca. 10 8		203, 204
Morphine	MnO4 -H	1	10 10	194
Naphthols	MnO4 -H	5	10 7a	205
Tetracycline	Br2-OH	4	10 5	206
	Dibromodimethyl hydantoin	1	10 6	207
Sulphenyl esters	Iodosobenzene	5	10 4	208
Loprazolam	MnO4 -H	7	10 6	209
Streptomycin	MnO4 -H	ca. 10 5		210
Sulfite	MnO4 -H	1	10 6a	211
	MnO4 -H	1.5 10 8b		212

aWith sensitizer.

bWith 3-cyclohexylaminopropanesulfonic acid.

loprazolam [196]. In these cases, the sample containing the drug was injected into a phosphoric acid stream that was subsequently merged with the oxidant stream, offering an extremely simple, economic, and effective experimental procedure. Since then, a wide range of such reactions have been reported mainly in the analysis of drugs [194, 197–212]. Table 3 shows some of the first direct CL determinations.

5. CONCLUSIONS

General books [213–217], chapters [218], and reviews were published in the 1980s reporting the suitability of CL and BL in chemical analysis [219–222], the specific analytical applications of BL [223], the CL detection systems in the gas phase [224], in chromatography [225, 226], the use of different chemiluminescent tags in immunoassay, and applications in clinical chemistry [227–232] as well as the applications of CL reactions in biomedical analysis [233].

In the 1990s, CL detection was coupled as a detection system in the by then recently introduced separation technique in routine analysis, capillary electrophoresis (CE). Hara’s group reported the first application in 1991 [234], using

Historical Evolution of Chemiluminescence

33

a homemade CE-CL device for the determination of proteins using the PO-CL reaction. Since then, more investigations have been carried out to improve the detection devices and their coupling to the flowing stream as well as to optimize the different experimental factors influencing both detection and separation processes.

It is clear from the literature, and as stressed on various occasions, that CL measuring systems applied to detection in flowing streams and immunoassays are of growing importance, based on the advantages including low detection limits and the relatively simple instrumentation. Also, CL-based detection appears to be of utmost importance in the area of CE micromachining in which micro- channel-based mixing of reagents and the creation of reaction chambers open new analytical perspectives. It should be emphasized as well that the fast evolution of immobilization techniques (e.g., employing beds of CL reagents or enzymes) improves the application of CL detection, among others, in separational streams and in the biosensing area.

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