ANALYTICA

CHIMICA
ACTA
ELSEVIER Analytica Chimica Acta 327 (1996) 161-165

Hydrogen peroxide assay by using enhanced chemiluminescence

of the luminol-H202--horseradish peroxidase system:
Comparative studies
A. Navas Diaz, F. Garcia Sanchez*, J.A. Gonziilez Garcia
Department of Analytical Chemistry, Faculty of Sciences, University of Malaga, 29071 Malaga, Spain

Received 20 July 1995; accepted 23 January 1996

Abstract

p-Iodophenol, p-coumaric acid, phenol and aniline are enhancers of the luminol-H202-horseradish peroxidase
chemiluminescence. These enhanced and unenhanced chemiluminescences were used to assay low concentrations of
hydrogen peroxide. The curves of cherniluminescence intensity maxima against hydrogen peroxide concentration were fitted
to third order polynomial regressions, which permit a very good approach to the experimental results. Double the sensitivity
(RSD 5 4.3%) and a slightly lower detection limit (e.g. 0.08 pM with p-coumaric acid of 0.18 pM without enhancer) were
obtained with enhanced chemiluminescence than with unenhanced chemiluminescence.

Keywords: Enhanced chemiluminescence;Luminol; Hydrogen peroxide; Horseradishperoxidase

1. Introduction peroxide concentration between lo-’ and 10e4M;

with copper CL is not linearly proportional to
Numerous methods have been proposed for the hydrogen peroxide; with peroxidase the CL is
detection and determination of low concentration of proportional to the square of the hydrogen peroxide
hydrogen peroxide. The most sensitive are the concentration. In this paper, we centered our attention
chemiluminescence-based methods, by using perox- in the luminol-H202-horseradish peroxidase system.
yoxalates, acridinium phenyl carboxylates, luminol, This system extends the pH range of luminol
etc. The chemiluminescence of luminol catalyzed by chemiluminescence to lower values.
different catalysts or co-oxidants has often been used The chemiluminescence of the luminol-H202-
in hydrogen peroxide assays. The relation between horseradish peroxidase system have been used to
the concentration of hydrogen peroxide and the assay peroxidase and conjugates of this enzyme [2],
chemiluminescence (CL) is related to the catalyst enhancers [3] or inhibitors [4] of this chemilumines-
or co-oxidant used [I]. With hexacyanoferrate(III) cence, and also hydrogen peroxide and other
the CL is linearity proportional to the hydrogen compounds that produce hydrogen peroxide for the
luminol-H202-horseradish peroxidase reaction in
coupled reactions [5].
* Correspondingauthor. Fax: +34 52 131884.

ooO3-2670/96/$15.000 1996 Elsevier Science B.V.All rights reserved

PII SOOO3-2670(96)00077-3
162 A. Navas Diaz et al./Analytica Chimica Acta 327 (1996) 161-165

On the other hand, the chemiluminescence of this luminescence spectrometer controlled by the FLDk
system enhanced by p-iodophenol, p-coumaric acid, (FL Data Manager, from Perkin-Elmer) program
firefly luciferin, etc. has commonly been applied to with the light source switched off. The apparattt
immunoassay and determinations of peroxidase and was set in the phosphorescence mode with O.OOm!
peroxidase conjugates [6-81, enzymatic assay by delay-time and 120ms gate time. The slit-width
using pro-enhancers or pro-anti-enhancers of this of the emission monochromator was set at 20nn
system [9,10], and in determinations of anti- with X(em) = 425 nm and the photomultiplie
enhancers [ 111. Also, the chemiluminescence of the voltage set to 900V. The samples were placed in ;
system enhanced by p-iodophenol was applied to quartz cuvette continuously stirred with a magnetic
hydrogen peroxidase determination [ 12,131. stirrer.
This paper describes a comparative study of the Polynomial fits were made with the Leatherbarrow
determination of hydrogen peroxide by using the R.J. (1990) GraFit Version 2.0 (Erithacus Software)
chemiluminescence of the luminol-H202-horserad-
ish peroxidase system without enhancer, and en- 2.3. Procedure
hanced by phenol, aniline, p-coumaric acid and p-
iodophenol. In this study, different polynomial Solutions of 0.01 M luminol and 0.1 M hydrogen
regressions were used to fit the experimental results peroxide (and 0.001 M enhancer in enhancec
to a calibration graph. chemiluminescence) were added to a quartz cuvettc
with a 0.1 M tris-HCl buffer at pH 8.4. This mixture
was diluted and distilled and demineralized water to i
2. Experimental final volume of 3 ml. The solution was continuousl!
stirred with a magnetic stirrer. The chemiluminescen
2.1. Reagents reaction was triggered by manual injection of 1001.1
of a 37.14 U/ml horseradish peroxidase solution
All stock solutions were prepared in distilled and through a septum. The progress of light emission
demineralized water. Luminol (5-amino-2,3-dihydro- was monitored and recorded. Chemiluminescencc
1,4_phthalazinedione) (Sigma, St. Louis, MO, USA) intensity maxima were measured.
was prepared by dissolving 0.0913 g of luminol
(97%) in a few drops of dilute NaOH solution the
volume was adjusted to 50ml with u-is-HCl buffer 3. Results and discussion
0.1 M (pH 8.5). Horseradish peroxidase type VI-A
(Sigma) 73 U/ml was prepared in u-is-HCl buffer (pH 3.1. Optimization of parameters
8.5); hydrogen peroxide (Panreac Montplet and
Esteban S.A., Barcelona, Spain) was prepared from The concentrations of luminol and p-iodopheno
hydrogen peroxide 6% w/v by diluting with distilled (Fig. 1) were optimized to obtain the greates
and demineralized water to 0.1 M. chemiluminescence intensity maxima with ;
p-Coumaric acid (Chydroxycinnamic acid) was 0.67 pM hydrogen peroxide concentration (Fig. 1)
from Sigma; p-iodophenol was purchased from This luminol final concentration (66.7 @I) was usec
Aldrich-Chemie, Steinheim, Germany; phenol was in the chemiluminescence enhanced by phenol
supplied by Merck, Darmstadt, Germany; and aniline aniline, p-coumaric acid and for unenhanced chemi
was from Probus S.A. Badalona, Spain. All stock luminescence. All the measurements were made in ;
solutions of these compounds were 0.001 M in tris-HCl buffer at pH 8.4. We used the same optima
distilled and demineralized water. concentrations of phenol and aniline as in [14], il
which approximately the same concentrations o
2.2. Instruments luminol, horseradish peroxidase were used and pI
as in our present paper, but with a 2mM hydrogen
The chemiluminescence experiments were carried peroxide. p-Coumaric acid concentration was opti
out in a Pet&n-Elmer LS-50 (Beaconsfield, UK) mized with the same concentration of luminol
 A. Navas Diaz et al./Analytica Chimica Acta 327 (19%) 161-165 163

Table 1
Reduced &i-square value for each polynomial regression of the
Iirst, second or third order
Enhancer Order = 1 Order = 2 Order = 3

None 0.711 0.758 0.310

Phenol 0.671 0.780 0.691
Aniline 100.0 20.61 4.02
p-Coumaric acid 34.6 13.94 4.41
p-Iodophenol 1371 184.6 28.00

0 20 40 60 80

[plodophenol] pi%l
horseradish peroxidase, hydrogen peroxide and pH as
p-iodophenol (Fig. 1).
The final concentrations in the cuvette used in the
hydrogen peroxide assay were: luminol 66.67 PM,
horseradish peroxidase 1.24 U/ml, tris-HCl 0.033 M
at pH 8.4. Optimal concentrations for each enhancer
were: p-iodophenol 16.67 PM, p-coumaric acid
0.67 pM, phenol 0.1 mM and aniline 0.333n-M.
Hydrogen peroxide concentrations were measured
between 0 and 0.67 PM.

0 2 4 6 8
3.2. Polynomial jts
[p-Coumnric acid]bM
Table 1 shows the reduced chi-square values for
Fig. 1. Chemiluminescence intensity maxima vs. concentrations each of the polynomial fits. The third order
of p-iodophenol and p-coumaric acid. Experimental conditions:
polynomial regressions were better than polynomial
H202 0.67 @I; luminol 66.67 FM; horseradish peroxidase 1.24 U/
ml; tris-HCl 0.033 M at pH 8.4.
regressions of the second and first order. Fig. 2 shows
the calibration graphs obtained with the use of aniline
240 _ 240

160 - 160 - 160 -

0 2 4 6 8 0 4 6 8 0 2 4 6 8

[HZ021 PM [HZ021 PM [H202] PM

Fig. 2. Different polynomial fits for the chemiluminescence intensity maxima of luminol enhanced by aniline vs. hydrogen peroxide
concentration. First order polynomial fit (left), second order polynomial fit (center), third order polynomial fit (right). Experimental conditions:
luminol 66.67 uM; horseradish peroxidase 1.24 U/ml; tris-HCI 0.033 M at pH 8.4 and aniline 0.333 n&l.
164 A. Navas Diaz et al./Analytica Chimica Acta 327 (1996) 161-165

by using polynomial regressions of first, second and Table 2

Parameters of the third order polynomial fit for each enhanced a
third order. Linear fits were acceptable in all cases
and for unenhanced chemiluminescence. The polynomials have the
(correlation coefficients greater than 0.9887), but the form:y=dx3+cx2+bx+a
third order polynomial fit was better for low concen-
Enhancer d C b a
trations of hydrogen peroxide. Fig. 3 shows the
maximal intensities of chemiluminescence against None 0.0763 -0.690 3.44 0.700
Phenol 0.0590 -0.560 4.84 0.872
concentrations of hydrogen peroxide (in PM) for each
Aniline -0.539 7.36 8.47 3.49
enhanced chemiluminescence and for the unenhanced p-Coumruic acid -0.416 5.12 16.39 -0.629
chemiluminescence with their third order polynomial p-Iodophenol -1.346 20.32 10.58 0.173
regression curves. Table 2 shows the parameters of
the third order polynomial fit for each enhancer and
for the emission without enhancer. 3.3. Detection limit, sensitivity and precision

p-Caumaric acid Table 3 shows the detection limits for the

p-lodophenol
240 determination of hydrogen peroxide by using the
enhanced chemiluminescence, and the enhanced

lzl
600

160 chemiluminescence by p-iodophenol, phenol, aniline

400
and p-coumaric acid. The procedure used to
200
80
determine the detection limits is shows in Fig. 4.

0 IIZI
0
0 2 4 6 8 Table 3
0 2 4 6 8
Detection limits, sensitivities and precisions (RSD) of the non-
fH2021pM
PO21 PM
enhanced and enhanced chemiluminescences

Aniline
Enhancer Detection limit Sensitivity RSD
Phenol
(n = 3) (n = 3) (n = 3)
140/ (IW (PM) (%)
None 0.18 0.19a 8.7’
Phenol 0.19 O.Ogb 4.lb
Aniline 0.13 0.05b l.gb
p-Coumaric acid 0.08 o.05c 4.3’
p-Iodophenol 0.12 0.08” 2.0a
0 2 4 6 R
Sensitivities were calculated at ‘3.33 uM, b2.67 PM, ‘1.33 uM
[H202]pM [H202] 1tM Hz%.

CL I
lntensi5 r
17. Maxima

8
5.6

0 El
0 2 4 6 8

I Hz021 PM

Fig. 3. Chemiluminescence intensity maxima vs. hydrogen per-

oxide concentration for the different enhanced chemiltinescences
0 0.04 0.08 0.12 0.16
and for the unenhanced chemiluminescence. Experimental condi-
tions: luminol 66.67 PM; horseradish peroxidase 1.24 U/ml; tris- [Hz021 PM
HClO.033 M at pH 8.4. Optimal concentrations for each enhancer
were: p-iodophenol 16.67 pM; p-coumatic acid 0.67 pM; phenol Fig. 4. Scheme for measurement of detection limit by a constant
0.1 mM and aniline 0.333 mM. variance method.
 A. Navas Diaz et al./Analytica
We used a constant variance method similar to that
shown in [15], but with some differences. To
calculate this variance we take each experimental
value (between 0 and 0.6667 uM H202) and add its
standard deviation. The differences between experi-
mental results (with standard deviation added) and
calculated results by the third order polynomial
regression were measured. The greatest positive
difference between the experimental values (with
standard deviation added) and calculated values was
taken as the variance constant and added to the
calculated signal of the blank. The detection limit
was calculated from this signal by using the third
order polynomial regression.
Table 3 shows the sensitivities calculated for the
unenhanced chemiluminescence and for the chemi-
luminescence enhance by phenol, aniline, p-coumaric
acid and p-iodophenol. We calculated this sensitivity
by dividing the standard deviation of the intensity
maxima of a sample of hydrogen peroxide by the
average slope of the calibration graph. We take as the
average slope the slope of the linear fit.
Precisions (RSD) are also shown in Table 3. These
precisions were measured as relative standard
deviations (RSD) of the calculated concentrations of
the hydrogen peroxide by using the third order
polynomial regression.
Acknowledgements
We thank the Comisi6n Interministerial de Ciencia
y Technologia (Projects PB93-1006 and BI094-
0548) for financial support.
Chimica Acta 327 (1996) 161-165 16.5
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