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ORIGINAL RESEARCHantiradical compound (Fig. 2). This setting allows thequantitative determination of the antiradical capacityof compounds in the range of µmol/L and sub-µmol/Lconcentrations (33–36).The response of the hydrogel system to antiradicalcompounds is investigated by using Trolox, a stablewater-soluble analogue of α-tocopherol. This compoundis widely used as a reference in such assays and it isassumed that it is able to scavenge two radical species,resulting in the formation of quinoidal analogues (37–44). The area under the CL decay curve, indicating thetotal light emitted, also represents the total amount ofthe reactive species present in the reaction media which,ultimately, lead to light emission. Radical scavengingcompounds will consume these reactive species and suppressthe light emission until all antiradical compoundsin the system are completely depleted. The emissionsuppression area (S SUP = total emission in the absenceof antiradical—total emission in the presence of antiradical)is therefore directly proportional to the number ofradical species deactivated by the antiradical and can beutilized as a measure for the antiradical capacity of thecompound (20).Addition of the antiradical to the hydrogel containingluminol and hemin before adding hydrogen peroxideleads to a drastic change in the emission intensitiesand only slight variations in the kinetic emission profile(Fig. 4A). A linear correlation between Trolox concentrationand the total light inhibition is observed (Fig. 4B)with a regression equation of total light inhibition =0.01 + 0.20 [Trolox] (R = 0.9995, n = 5), demonstratingthat this system can be utilized for antiradical capacitydetermination in analogy to the luminol CL antiradicalassay in aqueous medium (20).An antiradical added to the system will compete withluminol for the hemin-derived oxidizing intermediatesand any other oxidant species present in the reactionmixture, as well as intercepting luminol-derived freeradical species and oxidants. In this way, a delay periodin CL emission would be expected until all antiradicalspresent have been consumed. This lag-time should beproportional to the antiradical concentration and itscapacity, as it results from the oxidation of a non-CLsubstrate instead of luminol (45). However, the additionof antiradicals prior to initiation of the radical reactionmay interfere in the initial radical formation step, whichcould lead to less reliable results than those obtainedwith addition of antiradicals during the reaction course,where a constant steady-state concentration of radicalsis already established (46). A delay phase is notobserved in the PVP hydrogel system; however, theemission intensities are considerably lower in the presence(I AOH ) than in the absence (I) of the antiradical. Alinear correlation between the light intensity inhibition(I − I AOH /I) and the concentration of Trolox is observed(Fig. 4C), showing the existence of a competitionE. L. Bastos et al.Figure 4. (A) Effect of Trolox addition on luminol CL, usingthe PVP hydrogel CL system. (B) Correlation of emissionsuppression areas (S SUP ) with the concentration of addedTrolox. (C) Correlation of the suppression of light intensity(I − I AOH /I) with the concentration of added Trolox. [Luminol],1.0 mmol/L; [hemin], 7.7 nmol/L; [H 2 O 2 ], 0.1 mmol/L; [PVP],80 mg/mL.Copyright © 2006 John Wiley & Sons, Ltd.Luminescence (In press)DOI: 10.1002/bio
Calibration analytical applications studies on PVP hydrogel-supported luminol CL ORIGINAL RESEARCH ORIGINAL RESEARCH7between the antiradical compound and luminol for theoxidizing species, e.g. Hem–Fe(IV) in the polymermatrix system (light intensity inhibition = 0.02 + 0.21[Trolox]; R = 0.9971, n = 5). This competition leads tolower emission intensities in steady-state conditions inthe presence of the antiradical and also to lower totalemission areas. Depending on the specific application,both parameters (suppression area and intensity inhibition)can be utilized to quantify antiradical capacity, asshown by the linear correlation between the two parametersand the Trolox concentration (Fig. 4B, C).The different behaviour of the PVP-supportedluminol CL system, as compared to analogous aqueousassays (20), is supposed to be related to diffusion factorsin the polymer system. One possible explanationcould be that the reaction rate might be determinedmuch more by the diffusion of the species than by itsreactivity, thereby ‘masking’ the much higher reactivityof the antiradical Trolox towards the oxidizing speciesand allowing the competition by luminol anion, even ifboth species are present in comparable concentrations.In fact, in the system studied the luminol concentrationis three orders of magnitude higher than the Troloxconcentration; however, in the analogous aqueoussystem Trolox is sufficiently more reactive and ableto scavenge all the oxidizing species present until itseffective complete depletion (20). Whatever may bethe exact reason for the different behaviour of theaqueous and PVP-supported luminol CL system in thepresence of an antiradical compound, it is shown in thiswork that the polymer-supported system can beperfectly utilized for antiradical capacity determinationand opens up the possibility of automation usingmicroplate luminometers.CONCLUSIONSIn conclusion, we have shown that the PVP hydrogel CLsystem can be utilized up to 90 days after preparation forthe determination of hydrogen peroxide concentrationand as an antiradical capacity assay, since optimizedconditions are used to maximize its sensibility and reproducibility.Furthermore, it can be conveniently utilizedfor the calibration of microplate luminometer PMTsand, consequently, the determination of CL quantumyields. This approach allows the direct comparison of theexperimental results (emission intensities and total lightemission) obtained in different research laboratories.AcknowledgementsA donation of 60% hydrogen peroxide by Solvay–Peróxidos do Brasil LTDA Co. is gratefully acknowledged.This work was supported by Fundação deAmparo à Pesquisa do Estado de São Paulo (FAPESP;Copyright © 2006 John Wiley & Sons, Ltd.98/05445-9, 00/06652-0, 01/07477-0), Conselho Nacionalde Desenvolvimento Científico e Tecnológico (CNPq),Coordenação de Aperfeiçoamento de Pessoal de NívelSuperior (CAPES) and Programa de Apoio ao DesenvolvimentoCientífico e Tecnológico (PADCT).REFERENCES1. Merényi G, Lind J, Eriksen TE. Luminol chemiluminescence—chemistry, excitation, emitter. J Biolumin. Chemilumin. 1990; 5:53–56.2. Rao PS, Luber JM Jr, Milonowicz J, Lalezari P, Mueller HS.Specificity of oxygen radical scavengers and assessment of thefree radical scavenger efficiency using luminol enhancedchemiluminescence. Biochem. 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Chemiluminescent chemicalsensors for oxygen and nitrogen dioxide. Anal. Chem. 1995; 67:2224–2230.9. Janasek D, Spohn U. An enzyme-modified chemiluminescencedetector for hydrogen peroxide and oxidase substrates. SensActuators B 1997; 39: 291–294.10. Collins GE, Rose-Pehrsson SL. Chemiluminescent chemicalsensors for inorganic and organic vapors. Sens. Actuators B 1996;34: 317–322.11. Bastos EL, Ciscato LFML, Bartoloni FH, Catalani LH, BaaderWJ. <strong>Studies</strong> on PVP hydrogel-supported luminol chemiluminescence:1. Kinetic and mechanistic aspects using multivariatefactorial analysis. Luminescence 2006; 21 (in press).12. Cotton ML, Dunford HB. <strong>Studies</strong> on horseradish peroxidase.XI. On the nature of compounds I and II as determined from thekinetics of the oxidation of ferrocyanide. Can. J. Chem. 1973; 51:582–587.13. Scolaro LM, Castriciano M, Romeo A, Patanè S, Cefali E,Allegrini M. Aggregation behaviour of protoporphyrin XI inaqueous solution: clear evidence of vesicle formation. J. Phys.Chem. B 2002; 106: 2453–2459.14. Rabek JF. Experimental Methods in Photochemistry andPhotophysics. Wiley: Bath, 1982.15. Lee J, Seliger HH. Spectral characteristics of excited states ofproduct of chemiluminescence of luminol. Photochem. Photobiol.1970; 11: 247–258.16. Lee J. Spectroscopic characterizations of luminol chemiluminescence.Appl. Spectrosc. 1971; 25: 142.17. Lee J, Seliger HH. Quantum yields of luminol chemiluminescencereaction in aqueous and aprotic solvents. Photochem. Photobiol.1972; 15: 227–237.18. Baader WJ, Stevani CV, Bastos EL. Chemiluminescence oforganic peroxides. In The Chemistry of Peroxides, Rappoport Z(ed.). Wiley: Chichester, 2006; 1211–1278.19. O’Kane DJ, Lee J. Absolute calibration of luminometers with lowlevellight standards. Methods Enzymol. 2000; 305: 87–96.20. Bastos EL, Romoff P, Eckert CR, Baader WJ. 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