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ORIGINAL RESEARCHsolution (10 mmol/L) was prepared in 1 mol/L NaOH,kept at 4°C and used within 10 days. The workingstock solution was prepared by dilution in phosphatebuffer (0.1 mol/L, Na 3 PO 4 /Na 2 HPO 4 ), pH 11.6. Thefinal luminol concentration was determined spectrophotometricallyat 347 nm (ε = 7600 mol/L/cm).Hydrogen peroxide (Peróxidos do Brasil, Brazil)was obtained as a 60% w/w unstabilized aqueoussolution. The final concentration after dilution withdemineralized water (18 MΩ, Milli-Q, Millipore) wasdetermined spectrophotometrically, as described byCotton and Dunford (12). Hemin (ferriprotoporphyrinIX chloride) was purchased from Sigma (St. Louis,USA). A stock solution was prepared by dissolving2.5 mg hemin in 5 mL 1 mol/L aqueous NaOH. Theworking solution is a 1:100 dilution with 1 mol/L NaOH(8 µmol/L). The concentration was determined spectrophotometricallyusing ε = 58400 mol/L/cm at 382 nm(13). Poly(N-vinyl-2-pyrrolidone), known as PlasdoneK-90 (M w = 1.2· × 10 6 ; M n = 3.6· × 10 5 ), was obtained fromGAF Chem. Co. (USA). Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was obtainedfrom Aldrich (Milwaukee, USA) and used as antiradicalstandard.InstrumentsThe CL emission experiments were performed on aBerthold EG&G 96V Lumimat microplate luminometer.Absorption spectra were recorded using a ShimadzuMultispec 1500 UV-visible spectrophotometer.Preparation of PVP hydrogel containing luminoland heminAliquots (200 µL) of a solution containing 120 mg/mLPVP and adequate concentrations of luminol and heminin phosphate buffer (pH 11.6, µ = 0.1 mol/L) were placedin a 96-well microplate, which was positioned 20 mm distantfrom a pen-type Heraeus (Hanau, Germany) 20 Wlow-pressure Hg lamp (λ em = 254 nm; 200 mm long). Thesolution was irradiated for 30 min under a slow flux ofinert gas. The resulting microplates can be used directlyor dried for storage and continuous use. For storage, themicroplates were submitted to a slow argon flux until athin film (ca. 2 mm) was formed, packed in PVC film andstored at 4°C.Hydrogen peroxide determinationThe freshly prepared 96-well microplate containing thePVP hydrogel-supported luminol–hemin system (PVPhydrogel CL system) was put into the cavity of aluminometer and thermostated to 25.0 ± 0.5°C. The reactionwas initiated by automatic jet injection of 100 µLaqueous hydrogen peroxide solution in adequateCopyright © 2006 John Wiley & Sons, Ltd.E. L. Bastos et al.concentration. To perform the experiments with storedmicroplates, 200 µL phosphate buffer (pH 11.6, µ =0.1 mol/L) were added to each well prior to placing itinto the luminometer cavity, followed by hydrogen peroxideaddition (100 µL) to initiate the measurement.The results obtained in both cases were very similar andfinal reagent concentrations were reported for a finalvolume of 300 µL (final concentrations: PVP, 80 mg/mL;luminol, 1.0 mmol/L; hemin, 7.7 nmol/L). Emission decaywas monitored for 1 h in cycles of 60 measurements/well/h.Quantification of the antiradical effect ofTroloxAliquots (60 µL) of a Trolox solution in adequateconcentration were added to each well of a microplatecontaining 200 µL PVP hydrogel CL system. Thereaction was initiated by addition of 40 µL aqueous0.75 mmol/L hydrogen peroxide solution to eachwell, using the automatic jet injection system presentin the Berthold luminometer (final concentrations:PVP, 80 mg/mL; luminol, 1.0 mmol/L; hemin, 7.7 nmol/L; H 2 O 2 , 0.10 mmol/L). Emission decay was monitoredfor 1 h in cycles of 60 measurements/well/h.Microplate luminometer calibrationAqueous media calibration. To a single well of a white96-well microplate thermostated to 25.0 ± 0.5°C, containing200 µL luminol solution (15.0 nmol/L) in phosphatebuffer (pH 11.6, µ = 0.1 mol/L), 40 µL 75.0 µmol/Laqueous hydrogen peroxide solution were added byautomatic injection. The reaction was initiated byaddition of 20 µL hemin solution (0.8 nmol/L). Aftercomplete emission decay in about 20 s, 20 µL hemin(2.4 nmol/L) solution were added and a low-intensityemission was observed. Finally, an additional injectionof the concentrated hemin solution was made (20 µLhemin 2.4 nmol/L), normally not leading to measurablelight emission, to assure complete luminol consumption.The final reaction mixture contains 0.37 nmol/L hemin,10.0 nmol/L luminol and 10.0 µmol/L hydrogen peroxidein 300 µL final volume.Hydrogel calibration. To a single well of a microplatefilled with 240 µL of PVP hydrogel CL system([luminol], 12.5 nmol/L; [hemin], 0.26 nmol/L), 40 µL75.0 µmol/L hydrogen peroxide solution were addedby automatic injection. After total emission depletion,an additional volume of 20 µL hemin (2.4 nmol/L)solution was added to the reaction mixture. In theexperimental conditions utilized, the last hemin additionleads to very low-intensity light emission and theemission area was, in all cases tested, less than 5% ofthe initial emission area. The final reaction mixtureLuminescence (In press)DOI: 10.1002/bio
Calibration analytical applications studies on PVP hydrogel-supported luminol CL ORIGINAL RESEARCH ORIGINAL RESEARCH3contains 80 mg/mL PVP, 0.37 nmol/L hemin, 10.0 nmol/L luminol and 10.0 µmol/L hydrogen peroxide in 300 µLfinal volume.Calculation method. The area of emission can be convertedfrom arbitrary units (a.u.) to Einstein (E, ‘mols’of photons) using the luminol conversion factor ( f lum ),which is calculated by equation 1:flumCopyright © 2006 John Wiley & Sons, Ltd.lumnlum= φ [ Eau / . .](1)Slumwhere φ lum is the luminol quantum yield, n lum is thenumber of mols of luminol and S lum is the area under thecurve of emission, which is obtained by integration ofthe light emission intensity as a function of the reactiontime. The quantum yield for a certain CL orbioluminescence (BL) reaction (φ sample ) is obtained byequation 2:sample lum photoφ samplensample= S f f(2)where S sample is the area under the curve of the BL/CLreaction to be calibrated, f lum is the luminol factorobtained from equation 1, n sample is the number of molsof the limiting reagent, and f photo is the photomultipliertube (PMT) wavelength sensitivity factor. The latterfactor is required if light emission from the samplesystem occurs in a wavelength region different fromluminol emission (420 nm), since commercial PMTsshow wavelength-dependent sensibility. The relativesensibility of a specific PMT with the emission wavelengthis normally available from the equipment manufacturer.For the PVP hydrogel CL system this factor isf photo = 1, as its emission spectrum is identical to that ofthe aqueous system (11).The emission intensity (I, a.u./s) can also be calibratedand thus expressed as calibrated intensity (I c ) in E/susing f lum and f photo (equation 3). In this way, light emissionintensities can be reported in absolute values andtherefore experimental results obtained by differentacquisition equipments (i.e. luminometers, photoncounters and fluorimeters) in different research laboratoriesbecome comparable:I C [E/s] = I[a.u./s] × f lum [E/a.u.] × f photo (3)Statistical and mathematical analysisAll values were expressed as mean ± standard deviation(SD) of at least three independent experiments. All calculationswere performed using Origin 6.1 (OriginLabCorp., 2000).RESULTS AND DISCUSSIONLuminometer calibration using thePVP-supported luminol–hemin systemPrimary calibration of photomultipliers can be accessedby the use of a source of light radiance calibrated withreference to black body radiation (14). However, severalissues concerning the light emission intensity, refractiveindex discrepancies and, most importantly, geometricfactors make the use of this methodology difficult (14–17). To overcome these technical difficulties, secondarycalibration with reference to the CL oxidation of luminoleliminates the necessity for complex geometrical factorcalculations and is widely used (15–18). The typicalprocedures consist of the reaction of luminol with hydrogenperoxide in the presence of hemin, which has anabsolute CL emission quantum yield (φ CL ) of 0.0124in alkaline aqueous media (17–19). This value is independentfrom luminol concentration over a wide range(10 −9 –2.0 mmol/L) and the methods feasibility requiresan at least 100 times molar excess of hydrogen peroxideover luminol and successive additions of catalyst to guaranteecomplete luminol consumption (17–19).The amount of light produced by the conventionalaqueous calibration system is compared with that of thePVP hydrogel CL system obtained in the same experimentalconditions (i.e. identical concentrations ofreagents). The obtained results allow us to determine theabsolute light emission of the luminol–hemin–hydrogenperoxide reaction in the cross-linked polymer. Theluminol conversion factor is determined from theaqueous calibration system as f lum = 6.87 ± 0.21 × 10 −18E/a.u. (equation 1), and the emission quantum yield inthe PVP hydrogel system is φ CL = 2.39 ± 0.07 × 10 −3 E/mol(equation 2).The amount of luminol used in calibration (3.0× 10 −12 mol) is able to generate a total emission of7.16 ± 0.22 × 10 −15 E in the PVP hydrogel system.The calibrated initial emission intensity (I C ) in thestandard PVP hydrogel system is determined to beI C = 9.99 × 10 −14 E/s; absolute emission intensities are alsoreported here for the analytical applications of thesystem.Stability of the hydrogel CL systemTo access the stability of the PVP-supported luminol–hemin system, the amount of light produced in the presenceof hydrogen peroxide has been monitored for 3months. Microplates were stored at 4°C under argonatmosphere and nine experiments (three independentreplicates in three microplates) were performed every 10days (Fig. 1).The emission intensity and φ CL proved to beconstant for about 30 days. After this period, aLuminescence (In press)DOI: 10.1002/bio
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