Short Communication

SPECIAL ISSUE
Design of an Amperometric Biosensor for the Determination of
Biogenic Amines Using Screen Printed Carbon Working
Electrodes
Dietlind Telsnig,a Kurt Kalcher,b Andreas Leitner,a Astrid Ortner*a
a Institute of Pharmaceutical Sciences, Department of Pharmaceutical Chemistry, Karl-Franzens-University, Humboldtstraße 46, 8010
Graz, Austria
b Institute of Chemistry, Department of Analytical Chemistry, Karl-Franzens-University, Stremayrgasse 16, 8010 Graz, Austria

*e-mail: astrid.ortner@uni-graz.at
Received: July 19, 2012
Accepted: September 5, 2012
Published online: October 16, 2012

Abstract
An amperometric biosensor with pea seedling amine oxidase and screen printed carbon electrode modified with
MnO2 for determining biogenic amines (BAs) was designed. The bio-component was immobilized with Nafion solu-
tion. The enzymatically produced hydrogen peroxide was determined to quantify BAs. All measurements were per-
formed in flow injection analysis system with phosphate buffer (66 mM, pH 7.5) as mobile phase at + 400 mV vs.
Ag/AgCl. A linear correlation was found for cadaverine and putrescine from 1–50 mM, for tyramine and histamine
from 10–300 mM respectively. The limits of detection were determined to be 0.3 mM for cadaverine and putrescine
and 3.0 mM for tyramine and histamine. The sensor was used to quantify BAs in chicken meat samples.

Keywords: Amperometric biosensor, Pea seedling amine oxidase, Screen printed carbon electrode, Biogenic amines

DOI: 10.1002/elan.201200378

BAs are low molecular weight organic bases. They are
formed of amino acids during bacterial degradation such
as ripening, processing or putrefaction. When ingested in
high amounts they can have negative health effects, espe-
cially in sensitive people [1]. BAs are thermally stable
and are therefore important marker substances for the
freshness of food. According to the Commission Regula-
tion (EC) 2073/2005 the statutory limit of histamine is
fixed to 200 mg/kg in fresh fish. However no limitation is
given for other BAs.
Due to their great importance in food industry differ-
ent approaches (HPLC and GC methods) have been
made to quantify them [2–4].
Bio-sensing systems have proved to be very versatile
tools for the determination of BAs since they generate
a direct signal, allow fast analysis and do not require
a preceding sample preparation. Several publications are
dealing with bio-sensing systems to quantify BAs [4–7].
PSAO is an herbal diamine oxidase extracted from pea
seedlings. It shows high selectivity for cadaverine and pu-
trescine and mediocre selectivity for tyramine and hista-
mine. It catalyses the oxidative deamination of BAs ac-
cording to the following reaction equation:
RCH2 NH2 þ O2 þ H2 O ! RCHO þ NH3 þ H2 O2
PSAO is a homodimeric protein, containing a Cu2 + ion
and a covalently bound 2,4,5-trihydroxyphenylalanine
quinine (TPQ) at each active site. Due to its sensitivity
for the irreversible inhibitor semicarbazide PSAO belongs
to the group of semicarbazide sensitive amine oxidases
(SSAOs).
Hydrogen peroxide is well approved as electrochemical
detecting agent and excellent results have been obtained
using MnO2 as mediator [8].
Previous experiments in this group have shown that
BAs can be quantified in biological samples using PSAO
as bio-component immobilized with Nafion films without
interfering matrix effects [9]. The enzymatically produced
hydrogen peroxide was then detected with a MnO2 modi-
fied carbon paste electrode. In these former investigations
these electrodes were produced manually making them
a valuable tool during the development of a new analyti-
cal method. Their flexibility – which is desirable at this
point of development – can cause troubles with reprodu-
cibility (varying composition, surface characteristics etc.).
To overcome this drawback and to proceed with the opti-
mization of this PSAO biosensor, a more stable working
electrode was investigated – a SPCE which was produced
semi-automatically.
In the present work, a SPCE modified with 4 % MnO2
(m:m) and PSAO was optimized for the quantitative de-
termination of BAs. Amperometric measurements with
flow injection analysis (FIA) system were performed to

Electroanalysis 2013, 25, No. 1, 47 – 50  2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 47
 Short Communication D. Telsnig et al.
SPECIAL ISSUE

Fig. 1. Typical amperogram of cadaverine. Injection of 50, 10, 5, 3, 2, and 1 mM (n = 6–7); mobile phase Sørensen phosphate buffer
(66 mM, pH 7.5), flow rate 1.00 mL/min, working potential + 400 mV vs. Ag/AgCl, injection volume 20 mL.

Table 1. Analytical parameters of the investigated substrates cadaverine, putrescine, tyramine, and histamine.
Analyte Linear range (mM) Slope R2 LOD (mM) LOQ (mM)
Cadaverine 1–50 8.279 0.9999 0.3 1.0
Putrescine 1–50 5.949 0.9999 0.3 1.0
Tyramine 10–300 0.428 0.9998 3.0 10.0
Histamine 10–300 0.455 0.9807 3.0 10.0

obtain data about substrate selectivity, analytical parame-
ters, lifespan, and determination in complex, biological
matrix.
All measurements were performed with Sørensen phos-
phate buffer (66 mM, pH 7.5) as mobile phase due to its
physiological properties. Hydrogen peroxide was analysed
at + 400 mV vs. Ag/AgCl. All measurements were per-
formed with a flow rate of 1.00 mL/min. During the anal-
ysis 20 ml of the respective analyte were injected into the
carrier stream.
The obtained hydrogen peroxide signals were well
shaped without any tailing. Due to a very low noise, the
system proved to be very sensitive.
The designed sensor had excellent substrate selectivity
for cadaverine and putrescine. Both amines showed
a linear dependence of the signal on the concentration
from 1–50 mM with a correlation coefficient of R2 =
0.9999. The linear correlation was calculated to be I
(nA) = 8.28c (mM) + 2.10 for cadaverine and I (nA) =
5.95c (mM) + 3.25 for putrescine. Tyramine and histamine
showed a linear correlation from 10–300 mM with a corre-
lation coefficient of R2 = 0.9998 for tyramine and 0.9807
for histamine. The linear correlation was calculated to be
I (nA) = 0.43c (mM) + 2.66 for tyramine and I (nA) =
0.46c (mM) + 18.84 for histamine. Figure 1 shows a typical
amperogram of cadaverine.
Limits of detection (LOD) were determined as peak :
noise ratio of 3 : 1 and limits of quantification (LOQ)
were estimated as the three-fold of LOD. The LODs
were determined to be 0.3 mM for cadaverine and putres-
cine, and 3 mM for tyramine and histamine, whereas the
LOQs were estimated to be 1 mM for cadaverine and pu-
trescine and 10 mM for tyramine and histamine.
The analytical parameters for cadaverine, putrescine,
tyramine, and histamine can be seen in Table 1.
The varying sensitivity of the sensor for the respective
substrate can be quantified by considering the electrode
surface as well as the concentration of the analyte. The
results can be seen in Table 2. The different sensitivities
are due to the selectivity of the enzyme towards the sub-
strate.
Special attention was paid to the repeatability of the
results as well as the lifespan of the sensor. When not in
use the sensor was stored at room temperature in the de-
tector. The sensor could be used for up to 40 runs (about
1400 injections) quantifying all four substrates. During
the first two days a significant decrease in sensitivity
could be observed followed by a long, comparatively
stable period.
Table 2. Sensitivity of the PSAO biosensor for the substrates ca-
daverine, putrescine, tyramine, and histamine (n = 6).
Analyte Sensitivity (nA mM1 cm2) SD [a] (nA mM1 cm2)
Cadaverine 12.5 3.0
Putrescine 9.0 2.6
Tyramine 0.65 0.2
Histamine 1.0 0.3
[a] Standard deviation

48 www.electroanalysis.wiley-vch.de  2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Electroanalysis 2013, 25, No. 1, 47 – 50
Amperometric Biosensor for Determination of Biogenic Amines

SPECIAL ISSUE
Fig. 2. Decrease of the cadaverine sensitivity of the sensor over time; mobile phase Sørensen phosphate buffer (66 mM, pH 7.5),
flow rate 1.00 mL/min, working potential + 400 mV vs. Ag/AgCl, injection volume 20 mL.

Figure 2 shows the decrease of the sensitivity for cadav-
erine over a period of 15 days.
The repeatability and the reproducibility were deter-
mined measuring cadaverine 10 mM (medium range). The
standard deviation of the intraday repeatability was calcu-
lated to be 0.8 % (n = 6), whereas the standard deviation
of the interday repeatability was calculated to be 20 %
(n = 6). If calculated without the first day of an electro-
des lifespan, the interday repeatability was calculated to
be 15 %. The reproducibility was determined by measur-
ing cadaverine 10 mM on 4 different electrodes. The stan-
dard deviation of the reproducibility was calculated to be
22 % (n = 4).
The sensor was tested for its applicability in complex
matrix by using extracts of fresh chicken meat as sample.
Biological samples are known to contain different ratios
of BAs, such as cadaverine, putrescine, tyramine, and his-
tamine. The developed sensor determines the sum of the
BAs present in the investigated extracts and displays
them as cadaverine equivalents.
Thus the method was calibrated using the standard ad-
dition method by adding different amounts of cadaverine
to the extracts (n = 3).
Figure 3 shows the linear correlation calculated from
the obtained signals. The correlation coefficient was cal-
culated to be R2 = 0.9744.
The amount of BAs in the chicken meat sample was
calculated to be 124 mg/kg ( 5 mg/kg) cadaverine equiv-
alents. This result is comparable to those published for
food products (fish, pork, cheese) with values ranging
from 70–300 mg/kg [4, 10].
The designed PSAO biosensor proved to be a valuable
analytical tool for the determination of BAs. Due to the
optimization of the working electrode and the measure-
ment system, the linear range was broadened and the sen-
sitivity was improved significantly compared to earlier
studies [9]. Extensive studies of the electrodes lifespan
showed that one electrode could be used for up to
15 days and 1400 sample injections. The PSAO biosensor

Fig. 3. Determination of BAs in chicken meat via standard addition method (n = 3); mobile phase Sørensen phosphate buffer
(66 mM, pH 7.5), flow rate 1.00 mL/min, working potential + 400 mV vs. Ag/AgCl, injection volume 20 mL.

Electroanalysis 2013, 25, No. 1, 47 – 50  2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.electroanalysis.wiley-vch.de 49
 Short Communication
showed excellent results in complex matrix and is well
SPECIAL ISSUE
suitable for the determination of BAs in food.
Experimental
All used chemicals and reagents were of analytical grade.
PSAO EC 1.4.3.6 was a generous gift from Bio-Research
Product, Inc. Carbon ink (type C2030519P4) was pur-
chased from Gwent Electronic Materials Ltd. Manganese
dioxide (85–90 %), KH2PO4 (99 %), Na2HPO4·2H2O
(99.5 %), and ethanol (96 %) were obtained from Merck.
Nafion 5 % solution (m:m) (perfluorinated, ion exchange
resin), cadaverine dihydrochloride, tyramine hydrochlo-
ride, and histamine dihydrochloride (> 99 %) were pur-
chased from Sigma Aldrich. Glycerolum purum (> 98 %)
was purchased from Fluka Chemie AG. Putrescine, was
obtained from Acros Organics. Ceramic support plates
(sintered aluminum oxide) for the production of the
SPCEs were purchased from CoorsTek. The chicken meat
was purchased at a local supermarket.
All aqueous solutions were prepared with highly pure
water, freshly prepared in the lab by a cartridge system
(Milli-Q).
Sørensen phosphate buffer (66 mM, pH 7.5) was pre-
pared by mixing a KH2PO4 solution (66 mM) and
a Na2HPO4·2H2O solution (66 mM) until the required pH
was achieved. PSAO was dissolved in Sørensen phosphate
buffer with a concentration of 10 mg/mL aliquoted and
stored at 20 8C to avoid frequent freeze-thaw cycles. All
substrate solutions were prepared by dissolving the re-
agent in Sørensen phosphate buffer, stored at 4 8C and
prepared freshly every two days. Nafion was neutralized
with tetrabutylammonium-hydroxid 40 % (m:m) before
use.
All electrochemical measurements were carried out
with a Perkin Elmer/BAS Amperometric detector LC 4B
using a National Instruments 6120 AD converter and
a Gilson Minipuls 3 peristaltic pump with a Tygon R3607
tube (ID 1.3 mm, wall 0.86 mm). Data were analyzed
with LabView SignalExpress as well as PeakSimple soft-
ware. The interpretation of the collected data was per-
formed with MS Excel.
The printing of the working electrodes was performed
using a semi-automatic screen printer (MLP USA). An
Ag/AgCl electrode and a stainless steel electrode were
used as reference and auxiliary electrode respectively. A
spacer with a size of 3  22  0.254 mm was used to gener-
ate a measuring chamber. The electrochemical cell was
put into a Faraday cage to prevent parasitic signals.
50 www.electroanalysis.wiley-vch.de  2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
D. Telsnig et al.
The PSAO modified biosensor was prepared by drop-
coating 10 mL PSAO solution on the electrode surface
and allowing it to dry at room temperature. After that
the surface was covered with 10 mL Nafion solution in
two steps by drop-coating with 5 mL respectively and al-
lowing it to dry.
The determination of BAs in chicken meat samples was
performed according to Carelli et al. [10]. Briefly, chicken
meat was homogenized using a common household blen-
der shaft. 2.0 g meat was weighed in a centrifuge tube
and 5.0 mL Sørensen phosphate buffer were added. The
sample was homogenized by vortexing for 1 min. The
sample was extracted by ultrasonication for 10 min. at
room temperature using a Transsonic 660/H; Elma ultra-
sonication bath. After that the tube was centrifuged at
3500 rpm for 10 min at room temperature using a Haereus
Instruments Labofuge 400 (d = 110 mm) to separate the
residue from the liquid. The supernatant was transferred
to a 20 mL volumetric flask and the extraction was re-
peated two more times. The volumetric flask was filled up
to the mark with phosphate buffer and an aliquot was fil-
tered through a 0.22 mm SCFA filter.
Acknowledgement
The authors would like to thank Bio-research Product,
Inc. for providing the PSAO.
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