Journal of Biotechnology 119 (2005) 172–180

Amperometric biosensor for the detection of hydrogen peroxide

using catalase modified electrodes in polyacrylamide
Shailly Varma, Bo Mattiasson ∗
Department of Biotechnology, Lund University, Lund, Sweden

Received 5 November 2004; received in revised form 24 January 2005; accepted 29 January 2005

Abstract

A simple biosensor for the detection of hydrogen peroxide in organic solvents has been developed and coupled to a flow
injection analysis (FIA) system. Catalase was entrapped in polyacrylamide gel and placed on the surface of platinum (working
electrode) fixed in a Teflon holder with Ag-wire (auxiliary electrode), followed by addition of filter paper soaked in KCl. The
entrapped catalase gel was held on the electrode using membranes. The effects of cellulose and polytetrafluroethylene (PTFE)
membranes on the electrode response towards hydrogen peroxide have been studied. The modified electrode has been used to
study the detection of hydrogen peroxide in solvents like water, dimethyl sulfoxide (DMSO), and 1,4-dioxane using amperometric
techniques like cyclic voltammetry (CV) and FIA. The CV of modified catalase electrode showed a broad oxidation peak at
−150 mV and a clear reduction peak at −212 mV in the presence of hydrogen peroxide. Comparison of CV with hydrogen
peroxide in various solvents has been carried out. The electrode showed an irreversible kinetics with DMSO as the solvent.
A flow cell has been designed in order to carry on FIA studies to obtain calibration plots for hydrogen peroxide with the
modified electrode. The calibration plots in several solvents such as water, dimethyl sulfoxide, 1,4-dioxane have been obtained.
The throughput of the enzyme electrode was 10 injections per hour. Due to the presence of membrane the response time of the
electrode is concentration dependent.
© 2005 Elsevier B.V. All rights reserved.

Keywords: Catalase modified electrode; Hydrogen peroxide; Amperometry; Organic phase enzyme electrodes

1. Introduction analytical methods. Hydrogen peroxide can be moni-

tored directly at the surface of metal electrodes such as
Enzyme electrodes combine the specificity of an platinum polarized to 600 mV (Lingane and Lingane,
enzyme to an electrochemical system. They have been 1963). It can be monitored at low overpotentials ca.
used for the detection of various analyte molecules in 0 mV using enzyme modified electrodes or chemically
modified electrodes. Electrochemical based biosensors
∗ Corresponding author. Tel.: +46 46 2228264; for the detection of hydrogen peroxides in aqueous
fax: +46 46 2224713. media have been extensively studied using enzymes
E-mail address: [email protected] (B. Mattiasson). like horseradish peroxidase (Cass and Smit, 1992;

0168-1656/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.jbiotec.2005.01.020
 S. Varma, B. Mattiasson / Journal of Biotechnology 119 (2005) 172–180 173

Gorton et al., 1992, 1999; Wang et al., 1993; Xiao et al., organic peroxides such as cumene hydroperoxide or
1999), catalase (Varma and Mitra, 2002; Zhang et al., tert-butylhydroperoxides, etc. It was found that the
2002), etc. and inorganic complexes such as Prussian enzyme could also use dibenzoyl peroxide (Horozova
blue (Haghighi et al., 2004; Karyakin, 2001; Koncki, et al., 2002) as the substrate. The general mechanism
2002). There is interest in monitoring hydrogen perox- of action of catalase is as follows:
ide concentrations ranging from less than micromolars
H2 O2 + Cat-Fe(III) → H2 O + Cat-Fe(IV) O
in biological samples to several orders of magnitude Catalase (Compound-I)
higher in environmental or industrial samples. It is
possible to monitor electrochemically such a range of H2 O2 + Cat-Fe(IV) O → H2 O + O2 + Cat-Fe(III)
concentrations by suitably tailoring the enzyme immo-
bilization or the electrode surface. In the catalytic cycle hydrogen peroxide acts as
Organic phase enzyme electrodes (OPEEs) as ana- both donor as well as acceptor of electrons. It has
lytical biosensors based on the detection of hydrogen been shown that catalase can also show peroxidase
peroxide and other organic peroxides have been stud- activity by catalyzing the oxidation of molecules
ied. Mediated electron transfer in horseradish perox- such as ethanol, methanol, formic acid, quinones,
idase using ferrocene (Reviejo et al., 1994), as well etc. Catalase based amperometric sensors for the
as potassium hexacyanoferrate (Schubert et al., 1991, detection of ethanol (Akyilmaz and Dinckaya, 2003)
1992) have been investigated in organic solvents. OPEE and 3-chloroperoxibenzoic acid have been reported
as biosensor for the determination of tyrosine and per- (Horozova et al., 2002).
oxide in anti-infection formulations has been studied
(Wang et al., 1993). These biosensors have been used
for hydrogen peroxide determination in insoluble drug 2. Materials and methods
matrices; a small quantity of buffer solution was needed
for the biosensor to be operational. Catalase based 2.1. Materials
biosensor has been used to study the hydrogen per-
oxide detection in several organic solvents using deter- Catalase (C-1345-1927 U/mg), 30% hydrogen
gent dioctylsulfosuccinate (AOT)/toluene (Escobar et peroxide, cellulose membranes were procured from
al., 1990). Tomassetti et al. (Campanella et al., 1998, Sigma (St. Louis, USA). Polytetrafluoroethylene
2003) used catalase entrapped in kappa-carrageen gel (PTFE) membranes were purchased from Universal
to construct a biosensor for the detection of peroxides in sensors Inc., USA. Acrylamide/Bis acrylamide (40%),
several organic solvents and cosmetic samples. Direct N,N,N ,N -tetra-methyl-ethylenediamine (TEMED),
electron transfer using catalase modified electrodes has ammonium per sulfate (APS) were obtained from Bio
been demonstrated on glassy carbon (Varma and Mitra, Rad, CA, USA. All other chemicals like KH2 PO4 ,
2002), on pyrolytic graphite in polyacrylamide hydro- KCl, KOH, and HCl were of analytical grade obtained
gels (Lu et al., 2003). from Merck.
We were interested in the determination of hydrogen
peroxide in organic solvents. A suitable amperometric
biosensor system has been developed and coupled to 3. Methodology
an indigenously designed flow injection analysis (FIA)
setup. The biosensor was developed using catalase (EC: 3.1. Immobilization of catalase (entrapment)
1.11.1.6) as the model enzyme. Catalase is a tetrameric
enzyme, with a molecular weight of 240 kDa; it belongs One milligram catalase was entrapped in 1 ml
to the class of oxidoreductases (Eventoff et al., 1976). of 30% polyacrylamide gel (PAG). Acrylamide/bis-
The prosthetic group is protoporphyrin IX, the enzyme acrylamide (40% stock (3.75 ml)) in 0.875 ml of
has four heme centers. Bovine catalase has strongly Tris–HCl (pH 7) was deareated for 15 min, to this 5 mg
bound NADP, which probably has a protective role catalase in 1 ml Tris–HCl (pH 7) was added and swirled
to play. Catalase uses hydrogen peroxide as the sub- gently followed by the addition of 0.065 ml APS (1%)
strate nonetheless the enzyme can also act on other and 0.0025 ml TEMED. The gel was poured into the
174 S. Varma, B. Mattiasson / Journal of Biotechnology 119 (2005) 172–180

volume between clean glass plates with Teflon spacer an electrochemical cell of 10 ml working volume was
(0.25 mm) fixed on three edges of the glass plate and used. The electrodes were connected to potentiostat in
was allowed to polymerize for 30 min. The enzyme gel the standard way.
was cut into several small circles of 5 mm diameter.
The gel disks were dried in vacuum and stored at 4 ◦ C. 4.2. Flow injection analysis (FIA)
Prior to use the gel was hydrated in 10 mM KH2 PO4
(pH 7) containing 100 mM KCl. For FIA studies a home made flow cell of wall-jet
type was designed by us. The flow cell was made of
3.2. Enzyme assay three parts: (i) base made of Teflon; (ii) the middle
block made of acrylate; and (iii) an electrode holder
Catalase activity was assayed by measuring the (Fig. 1). The electrode was fitted into the cell by an
decrease in the absorbance of hydrogen peroxide at electrode holder. The electrode with the holder was
240 nm as a function of time. Catalase causes the fitted in the middle block and screwed into the Teflon
decomposition of hydrogen peroxide into water and base. The flow cell comprises of an inlet, outlet and
molecular oxygen. So a decrease in the absorbance an analyte chamber. The analyte chamber was defined
with the progress of enzymatic reaction is observed. using a viton O-ring. A two electrode setup comprising
Ten millimolar H2 O2 was prepared by adjusting the of Pt-electrode and Ag-wire was used (vide supra). All
absorbance between 0.550 and 0.552 using 50 mM FIA studies were done using 10 mM phosphate buffer
potassium phosphate buffer (pH 7). The reaction mix- (pH 7.5) with 100 mM KCl as the carrier stream. The
ture consisted of varying concentrations of catalase, electrolyte buffer was deaereated for ca. 1 h.
and a final concentraion of 9.66 mM H2 O2 in a total Peristaltic pump (OEM 400 F/DM2), power supply
volume of 3 ml. The absorbance was measured after and speed control unit were assembled together in a
30 s. The decrease in the absorbance is proportional to home made housing box. A two-way six port injec-
the catalase activity. tion unit (MX7900-000) from Rheodyne was installed
in the same home made box. Marprene tubing (i.d.
3.3. Enzyme electrode preparation 0.75 mm) was used to pump the carrier stream through
the peristaltic pump. A potentiostat from Zäta elec-
Pt-electrode (1.6 mm diameter) from Bio Analyt- tronic, Sweden was connected to the two electrode
ical Systems (BAS, West Lafeyette, USA) was used system for applying potential and measuring the cur-
as the working electrode. The electrode was polished rent.
using 0.5 ␮m alumina slurry on a wet polishing cloth
for 5 min followed by thorough rinsing with double
distilled water. 5. Results and discussion
The Pt-electrode was fitted into a Teflon holder
with Ag-wire around it. On the surface of Pt-electrode, The modified enzyme electrode was tested for the
entrapped catalase gel was placed followed by filter detection of hydrogen peroxide in several solvent sys-
paper soaked in 3.0 M KCl covering the enzyme gel and tems like water, DMSO and 1,4-dioxane. CV and FIA
the Ag-wire. Cellulose membrane and (or) PTFE mem- studies were carried out in order to study the electro-
brane were carefully placed on the filter paper without chemical and kinetic aspects of the modified enzyme
the formation of air bubbles. electrode.

5.1. CV studies
4. Instrumentation
To study the kinetics of the modified electrode
4.1. Cyclic voltammetry (CV) studies CV studies were carried out. The working elec-
trode consisted of Pt/catalase/cellulose and (or) PTFE
Electrochemical workstation (BAS CV 50W) was membrane. Effect of membranes on the CV in aque-
used in the present study. To carry on CV studies ous solution is shown in Fig. 2. The CV of an enzyme
 S. Varma, B. Mattiasson / Journal of Biotechnology 119 (2005) 172–180 175

Fig. 1. Schematic representation of the indigenously designed flow cell. The flow cell comprises of a base made of Teflon which houses the
inlet, outlet and the analyte solution chamber. The volume of the analyte is defined by a viton O-ring. The electrodes (working and the counter
electrodes) are inserted by fitting it into a jacket, which in turn can be screwed into the holder connecting to the base. For details of FIA setup
see text.

modified electrode in electrolyte alone is shown in oxidation peak at −150 mV and a clear reduction peak
curve (a) in Fig. 2, wherein only non faradaic capacitive at −212 mV in the presence of hydrogen peroxide was
currents can be observed. In the presence of substrate observed. The electrode exhibits a sluggish behavior
(5 mM H2 O2 in water) clear faradaic response can be in the presence of PTFE membrane clearly indicating
monitored as seen in the CVs curves (b) and (c) in a mass transport limited condition (curve (c) in Fig. 2)
Fig. 2. The CV shows unequal redox peaks, a broad when compared to cellulose membrane (curve (b) in
Fig. 2). The effect of scan rate on the CV was studied
(Fig. 3). A linear relation between the square root of
scan rate and the reduction current values (95% confi-
dence limit and r2 0.91) was obtained as seen in the inset
in Fig. 3. In the case of hydrogen peroxide in water no
significant shift in the peak potential due to the vary-
ing scan rates was observed which actually indicates
a reversible behavior. Ideally for a reversible electron
transfer the
Ep should be ca. 60 mV, but the elec-
trode processes are mass transport limited hence there
is a large difference between anodic and cathodic peaks
positions (
Ep ) of ca. 300 mV (Table 1). The substrate
has to diffuse from the bulk to the electrode surface;
the gel thickness, degree of crosslinking and the mem-
brane contribute to the mass transport barriers, which
the substrate has to overcome before reaching the sur-
Fig. 2. Cyclic voltammetry of the two electrode setup: (a) in elec- face of the electrode. Similar behavior was observed
trolyte alone (non faradaic currents) and in the presence of 5 mM
H2 O2 ; (b) with cellulose membrane; (c) with PTFE membrane. The
in the case of hydrogen peroxide in dioxane. In the
electrolyte solution was 10 mM potassium phosphate (pH 7.5) in case of hydrogen peroxide in DMSO also the plot of
100 mM KCl, the scan rate used was 5 mV s−1 . square root of scan rate versus current was a linear
176 S. Varma, B. Mattiasson / Journal of Biotechnology 119 (2005) 172–180

Scheme 1. Proposed scheme of electron transfer at the modified

enzyme electrode surface.

electron transfer) as seen in Scheme 1. Usually the elec-

trochemical electron transfer is the rate limiting step
Fig. 3. Cyclic voltammetry of Pt/catalase/cellulose electrode, in the and plays a crucial role in the electron transfer kinetics
presence of 5 mM hydrogen peroxide in 10 mM phosphate buffer (irreversible or quasi reversible situations).
(pH 7.5) and 100 mM KCl. The CV shows an oxidation peak at
The catalytic efficiency of the modified electrode
ca. 150 mV and a clear reduction peak at −212 mV. There is no
significant change in the peak potential due to changing scan rate expressed as a ratio of reduction currents in the pres-
in the case of aqueous medium. The inset shows the linear behavior ence (ic ) and absence (id ) of substrate is shown in Fig. 4.
of current as a function of square root of scan rates for hydrogen A decrease in the (ic /id ) was observed with an increase
peroxide in different solvents. in the scan rate, which is a characteristic of electro-
chemical catalysis (Andrieux et al., 1980). Catalytic
curve (95% confidence limit and r2 0.955) as seen in efficiency increases with the increase in the concentra-
the inset Fig. 3. But from Table 1 it can be clearly seen tion of the substrate.
that the
Ep varies significantly with the scan rate.
A linear relation between the natural logarithm of scan 5.2. FIA studies
rate and the peak position (confidence limit 95% and r2
0.995) was observed, clearly indicating an irreversible FIA parameters were standardized using hydrogen
behavior (Bard and Falkner, 1980). peroxide in aqueous media, the carrier buffer was
At the electrode surface there are two processes 10 mM potassium phosphate buffer (pH 7.5) containing
occurring, firstly the electron has to be transferred from 100 mM KCl. An increase in the flow injection peaks
the enzyme to the substrate to form the product dioxy- with the change in potential from −100 to −600 mV
gen (enzymatic electron transfer), secondly the elec- was observed. From the CV studies, it can be seen that
trode reduces the product synthesized (electrochemical the reduction peak is observed at ca. −212 mV, hence

Table 1
The effect of scan rate ν (mV s−1 ) on cyclic voltammetric parameters, anodic current (ipa ), cathodic current (ipc ), difference in the anodic and
cathodic peak potentials (
Ep )
ν (mV s−1 ) H2 O2 in water H2 O2 in DMSO H2 O2 in 1,4-dioxane

ipa (␮A) ipc (␮A)
Ep (mV) ipa (␮A) ipc (␮A)
Ep (mV) ipa (␮A) ipc (␮A)
Ep (mV)
5 2.44 −8.86 −398 1.48 −7.27 −404 1.94 −8.95 −398
10 4.056 −11.35 −404 2.98 −9 −464 3.24 −11.45 −425
20 5.31 −15.33 −416 4.55 −10.7 −514 4.66 −14.05 −445
50 6.52 −21 −419 7.45 −16.26 −606 5.76 −18.7 443
100 – −24 – −21.5 −22.57
Cyclic voltammograms were recorded in the presence of 5 mM H2 O2 in three different solvent systems. Pt/catalase/cellulose was the working
electrode.
 S. Varma, B. Mattiasson / Journal of Biotechnology 119 (2005) 172–180 177

Fig. 4. Graph showing the influence of catalytic efficiency (ic /id ) of Fig. 6. Graph showing the calibration curve of enzyme modified
catalase modified electrode in 10 mM phosphate buffer (pH 7.5) and electrode to hydrogen peroxide. Solid lines represent the enzyme
100 mM KCl, with 5 mM hydrogen peroxide. electrode in response in the presence of cellulose and the dashed
lines in the presence of PTFE membrane. Each point is an average
of three peaks and the error bars represent the data obtained for three
a higher negative potential is suitable to maintain the different electrodes. The bias potential was −600 mV, flow rate was
enzyme in reduced state for obtaining efficient elec- 0.18 ml min−1 , sample size was 50 ␮l. The carrier stream was 10 mM
trocatalysis. An optimum potential of −600 mV was phosphate buffer (pH 7.5) with 100 mM KCl.
chosen for further studies.
The effect of flow rate on the electrode response Fig. 6 shows the calibration curve for hydrogen
towards 5 mM H2 O2 was studied (Fig. 5); with the peroxide in different solvents. Maximal response was
decrease in the flow rate an increase in the current observed in the case of aqueous solution. The effect
response was observed. This behavior is due to the fact of different membranes on the electrode response for
that, as the flow rate decreases the electroactive species hydrogen peroxide is tabulated in Table 2. Due to mass
is exposed to the electrode for a longer time hence a transport limiting conditions the apparent Km of sub-
higher current is observed. strate increases for the immobilized enzyme rendering
it possible to monitor high concentrations of hydro-
gen peroxide (mM). When coupled with a technique
like FIA it is possible to monitor even higher con-
centrations of the substrate. The linear concentration
range in the case of hydrogen peroxide in water was

Table 2
FIA results with different membranes in the electrode assembly when
used in three different solvent systems
H2 O2 in Sensitivity r2 Dynamic linear
(␮A mM−1 ) range (mM)
Pt/catalase/cellulose
Water 0.01762 0.99 0.5–100
DMSO 0.0024 0.982 10–75
1,4-Dioxane 0.0035 0.96 10–75
Pt/catalase/cellulose/PTFE
Fig. 5. Response of Pt/catalase/cellulose membrane as a function of
Water 0.0043 0.98 1–500
flow rate. The substrate pulses contained 5 mM H2 O2 , the sample
DMSO 0.00105 0.996 10–100
size was 50 ␮l; the bias potential was −600 mV and the electrolyte
1,4-Dioxane 0.00108 0.99 10–100
used was 10 mM phosphate buffer (pH 7.5) and 100 mM KCl.
178 S. Varma, B. Mattiasson / Journal of Biotechnology 119 (2005) 172–180

0.5–100 mM (Table 2). The response time of the elec- electron transfer if it is present as a thin monolayer
trode depends on the concentration of the hydrogen at the electrode surface. In electrochemical measure-
peroxide. In the case of DMSO and 1,4-dioxane a lin- ments the heme center of the enzyme can communicate
ear range 10–75 mM is observed. In the presence of with the electrode if the enzyme is suitably immobi-
cellulose membrane the ratio of 10 mM H2 O2 signals lized. Covalent coupling of catalase on glassy carbon
were 1:0.18:0.118 for water, 1,4-dioxane and DMSO, matrix using a 16 atom spacer arm could show a direct
respectively. The decreased sensitivity in the case of electron transfer (Varma and Mitra, 2002). In a similar
organic solvents could be due to the inactivation of the study the entrapment of catalase within polyacrylamide
enzyme. It has been reported that catalase was inactived matrix, the catalase could show a direct electron trans-
by dioxane in phosphate buffer (pH 7) using electro- fer when the gel was polymerized on glassy carbon
chemical studies as well as spectroscopic studies like (Lu et al., 2003). The direct electron transfer involves
CD and IR (Joo et al., 1996). Nearly 80% decrease only the few catalase molecules near the electrode
in the activity of free enzyme in 50% dioxane was surface.
observed and the decrease was proportional with the The proposed scheme of electron transfer reaction
increase in the dioxane:phosphate buffer ratio. Such an for the modified electrode is shown in Scheme 1.
inactivation could be inhibited by treating the enzyme Catalase causes the dismutation of hydrogen perox-
with polyethylene glycol. From our previous studies ide to water and dioxygen. In the native state the
on glutamate oxidase/Prussian blue on glassy carbon an enzyme is in Cat-Fe(III) (ferric state), which is oxi-
increase was observed in the sensitivity of the response dized to compound I represented as Cat-Fe(IV) (ferryl
towards substrates as glutamate and ␤-N-oxalyl-␣,␤- state) in the presence of hydrogen peroxide (Chance
diaminopropionic acid (␤-ODAP) as substrates when and Oshino, 1973; Jouve et al., 1997; Kirm et al.,
polyethyleneimine (PEI) was added in low concentra- 2002). The compound-I reacts with another molecule
tions (0.01–0.001%) (unpublished work). The stability of hydrogen peroxide to regenerate the native enzyme
is expected to be due to the hydrogen bond formation back with the release of water and dioxygen. The
between the protein and the PEI. electrode polarized at a suitable potential can detect
The use of PTFE membrane makes it possible to the oxygen released. Thus the electrode response is
monitor even higher concentrations of hydrogen per- directly proportional to the concentration of hydrogen
oxide (Table 2). The signal intensities were much lower peroxide.
as the membrane is hydrophobic in nature. A lin-
ear relationship between concentration of hydrogen
peroxide and the response was observed for a range 6. Conclusions
10–500 mM.
The stability of Pt/catalase/cellulose modified elec- We have developed a simple biosensor using cata-
trode was studied and 100% signal was retained up to lase to monitor hydrogen peroxide in aqueous as well
5 h of continuous 10 mM H2 O2 injections (figure not as organic solvents. Catalase was entrapped in 30%
shown). At a flow rate of 0.18 ml min−1 , the throughput polyacrylamide gel of 5 mm diameter. The catalase
of the sensor was 10 injections per hour. gel was placed on Pt-electrode (working electrode).
Catalase is one of the most efficient enzymes with a A two electrode setup has been developed. Effects of
turn over number of (200,000 molecules s−1 ), catalyz- membranes like cellulose and PTFE have been inves-
ing the dismutation of hydrogen peroxide into water tigated using CV and FIA studies. Kinetics of catalase
and molecular oxygen. It is a tetrameric enzyme; the modified electrode was studied using CV. From the
prosthetic groups are deeply buried within the protein CV studies, it is evident that the enzyme reaction is a
core ca. 20 Å below the molecular surface and ca. 23 Å diffusion-controlled process, the behavior is evidently
away from the tetrameric center. As a development irreversible in the case of DMSO.
towards the third generation biosensors, direct electron The modified electrode was coupled to a home
transfer in catalase has been shown by several research made FIA setup. Response of the modified electrode
groups (Liu et al., 2004; Lu et al., 2003; Varma and was thoroughly studied and standardized in aqueous
Mitra, 2002). The enzyme is capable of showing direct media and compared in organic solvents like DMSO
 S. Varma, B. Mattiasson / Journal of Biotechnology 119 (2005) 172–180 179

and 1,4-dioxane. The sensor could monitor very high Escobar, L., Salvador, C., Contreras, M., Escamilla, J.E., 1990. On
ranges of hydrogen peroxide (mM) though the response the application of the Clark oxygen electrode to the study of
enzyme kinetics in apolar solvents: the catalase reaction. Anal.
time depends on the concentration of the substrate as
Biochem. 184, 139–144.
the system is mass transport limited. Eventoff, W., Tanaka, N., Rossmann, M.G., 1976. Crystalline bovine
Monitoring hydrogen peroxides finds applications liver catalase. J. Mol. Biol. 103, 799–801.
in several fields like biotechnology, medical applica- Gorton, L., Joensson-Pettersson, G., Csöregi, E., Johansson, K.,
tions, food technology, cosmetics, etc. One finds appli- Dominguez, E., Marko-Varga, G., 1992. Amperometric biosen-
sors based on an apparent direct electron transfer between elec-
cation of hydrogen peroxide determination in several
trodes and immobilized peroxidases. Analyst 117, 1235–1241.
industrial processes such as phenolic polymerization, Gorton, L., Lindgren, A., Larsson, T., Munteanu, F.D., Ruzgas,
textile bleaching, paper pulping, and food sterilization T., Gazaryan, I., 1999. Direct electron transfer between heme-
where the detection as well as the control of hydro- containing enzymes and electrodes as basis for third generation
gen peroxide concentration are important parameters biosensors. Anal. Chim. Acta 400, 91–108.
in the process. The developed amperometric biosen- Haghighi, B., Varma, S., Alizadeh, F.M., Yigzaw, Y., Gorton, L.,
2004. Prussian blue modified glassy carbon electrodes—study
sor coupled with FIA setup shall be integrated to a on operational stability and its application as a sucrose biosensor.
process control for determination of hydrogen per- Talanta 64, 3–12.
oxide and controlling its concentration in synthesis Horozova, E., Dimcheva, N., Jordanova, Z., 2002. Study of catalase
reaction. electrode for organic peroxides assays. Bioelectrochemistry 58,
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Joo, H., Yoo, Y.J., Ryu, D.D.Y., 1996. A biosensor stabilized by
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Acknowledgments
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Jouve, H.M., Andreoletti, P., Gouet, P., Hajdu, J., Gagnon, J., 1997.
The work is a part of the GREENCHEM program Structural analysis of compound I in hemoproteins: study on
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