Studies on PVP hydrogel-supported luminol ... - ResearchGate

Calibration analytical applications studies on PVP hydrogel-supported luminol CL ORIGINAL RESEARCH ORIGINAL RESEARCH5of 6.8 × 10 −7 mol/L. The regression equation for thelinear dependence is S C = 7.78 × 10 −11 + 1.05 × 10 −5 [H 2 O 2 ]with correlation coefficient (R) = 0.9977 (n = 6). Athigher peroxide concentrations the total light emissionshows a significant decrease (Fig. 3A, insert), a factwhich may indicate the destruction of hemin by excessof peroxide and incomplete conversion of hydrogenperoxide. Similar results are obtained if the initial emissionintensities are correlated with [H 2 O 2 ]; in this casethe regression equation for the linear dependence isI C = 9.04 × 10 −16 + 5.83 × 10 −10 [H 2 O 2 ] with R = 0.9998(n = 6). Also the light intensities show a sharp decreaseat higher peroxide concentrations (Fig. 3B).As can be seen from Table 1, the observed detectionlimit (6.8 × 10 −7 mol/L) is compatible with several otherdetection methods based on luminol CL, immobilized orin solution (21–29).We also can predict from the MFA that the linearrange of the peroxide dependence can be extended tohigher concentrations by using higher hemin concentrations,without significant changes in the detection limit(11). The results obtained for the detection of hydrogenperoxide are very promising in terms of application automation.For example, oxidases could be inserted intothe polymeric matrix together with luminol and hemin,allowing the automatic detection of specific substrates,such as glucose or lactose, in the µmol/L concentrationrange by quantification of hydrogen peroxide generatedby enzyme catalysed substrate oxidation (30, 31).Figure 3. Correlation of total light emission (A) and emissionintensity (B) with the hydrogen peroxide concentration.[Luminol], 1.0 mmol/L; [hemin], 7.7 nmol/L; [PVP], 80 mg/mL.concentrations), since we are interested here in detectingH 2 O 2 at the low concentration range. A lineardependence of the calibrated total light emission (S C )with the concentration of H 2 O 2 is observed up to 5 ×10 −4 mol/L (Fig. 3A), with an estimated detection limitAntiradical assay: effect of Trolox on emissionintensities and emission areasIn order to use the luminol CL as the basis ofan antiradical assay, the kinetics should consist in slowlight emission decay (32). This behaviour indicates aconstant, or nearly constant, production of free radicalintermediates derived from luminol, hemin and hydrogenperoxide. From our MFA we chose intermediateconcentrations of all the three reagents to access slowemission decay and a relatively slow consumption of theTable 1. Detection limits for hydrogen peroxide using several chemiluminescencemethodsDetection limitSystem (mol/L) ReferenceLuminol–KIO 4 3.0 × 10 −8 (21)Luminol–K 3 Fe(CN) 6 6.0 × 10 −6 (22)Luminol–HRP 8.0 × 10 −6 (24)Luminol–peroxidase 1.0 × 10 −8 (25)Luminol–Co(II) 5.0 × 10 −9 (26)Luminol–calixarene/Cu(II) 3.0 × 10 −6 (27)DTMC 4.0 × 10 −8 (28)Luminol–hemin 3.0 × 10 −9 (23)Luminol–hemin–PVP 6.8 × 10 −7 This workDTCM, 7-(4,6-Dichloro-1,3,5-triazinylamino)-4-methylcoumarin.Copyright © 2006 John Wiley & Sons, Ltd.Luminescence (In press)DOI: 10.1002/bio