Talanta 71 (2007) 312–317
Determination of phenolic acids using Trametes versicolor laccase
Dilek Odacia, Suna Timura, Nurdan Pazarlioglua, Maria Rita Monterealib,
Walter Vastarellab, Roberto Pillotonb,∗, Azmi Telefoncua
aEge University, Faculty of Science, Biochemistry Department, 35100 Bornova-Izmir, Turkey
bENEA, Via Anguillarese 301, SP061, Santa Maria di Galeria, 00060 Rome, Italy
Received 4 November 2005; received in revised form 6 March 2006; accepted 6 April 2006
Available online 12 June 2006
Two biosensors based on Trametes versicolor laccase (TvL) were developed for the determination of phenolic compounds. Commercial oxygen
electrode and ferrocene-modified screen-printed graphite electrodes were used for preparation of laccase biosensors. The systems were calibrated
acid for laccase immobilised on a commercial oxygen electrode and 2.0–30.0?M caffeic acid, 2.0–10.0?M ferulic acid, 4.0–30.0?M syringic
acid for laccase immobilised on ferrocene-modified screen-printed electrodes. Furthermore, optimal pH, temperature and thermal stability studies
were performed with the commercial oxygen electrode. Both electrodes were used for determination of a class of phenolic acids, achieving a cheap
and fast tool and an easy to be used procedure for screening real samples of human plasma.
© 2006 Elsevier B.V. All rights reserved.
Keywords: Phenolic acid; Laccase biosensor; Ferrocene; Screen-printed electrode
Phenolic acids and their derivatives are widely in plant
kingdom (legumes, cereals, and fruits), their by-products (tea,
cider, oil, wine, beverages) and medicinal plants [1,2]. Caffeic
acid (3,4-dihydroxycinnamic acid), ferulic acid (3-methoxy-
4-hydroxycinnamic acid), syringic acid (3,5-dimethoxy-4-
hydroxybenzoic acid) have some important antioxidant char-
acteristics for metabolism. Caffeic acid is a kind of phenolic
acid, which has been found to be pharmacologically active as
an antioxidant, antimutagenic, anticarcigenic agent, lipoxyge-
nase inhibitor and it also has antibacterial, antiinflamatory and
styptic activities [3,4]. Ferulic acid arises from the metabolism
of phenylalanine and tyrosine. It is the most abundant hydrox-
ycinnamic acid in the plant world and occurs primarily in seeds
and leaves both in its free form and covalently linked to lignin
and other biopolymers. The dehydrodimers of ferulic acid are
important structural components in the plant cell wall and serve
to enhance its rigidity and strength [5,6]. Due to its phenolic
E-mail address: email@example.com (R. Pilloton).
a resonance stabilized phenoxy radical, which accounts for its
potent antioxidant potential. UV absorption by ferulic acid cat-
alyzes stable phenoxy radical formation and thereby potentiates
its ability to terminate free radical chain reactions. By virtue
of effectively scavenging deleterious radicals and suppressing
radiation-induced oxidative reactions, ferulic acid may serve
an important antioxidant function in preserving physiological
integrity of cells exposed to both air and impinging UV radia-
tion. Similar photoprotection is afforded to skin by ferulic acid
dissolved in cosmetic lotions. Its addition to foods inhibits lipid
peroxidation and subsequent oxidative spoilage. By the same
mechanism, ferulic acid may protect against various inflamma-
on the antioxidant potential of ferulic acid . Recently, pheno-
and plant extracts by using HPLC with mass spectrometry, UV
or electrochemical detection [7–11], capillary electrophoresis
raphy , gas chromatography–mass spectrometry [15–18],
sor . Compared with other methods, biosensors have the
0039-9140/$ – see front matter © 2006 Elsevier B.V. All rights reserved.
D. Odaci et al. / Talanta 71 (2007) 312–317
Fig. 1. Typical laccase-catalysed reactions .
methods . Amperometric biosensors have been prepared as
mediated and unmediated systems. Many electrodes combined
with laccase have been developed for detection of phenolics
126.96.36.199) are multi-copper oxidase having Type 1, Type 2, and
lated from plant sources (e.g., lacquer, sycamore, and tobacco)
and are extracellular enzymes [35,36]. Very recently, it has been
in Fig. 1, laccases reduce oxygen directly to water in a four-
electron transfer step without intermediate formation of soluble
hydrogen peroxide in expense of one-electron oxidation of a
variety of substrates, e.g., phenolic compounds .
Instead of oxygen, mediator can be used as an electroac-
tive compound. Ferrocene and its derivatives have been used in
enzyme active site and electrode [38,39]. Phenolic acids are
oxidised by laccase. When ferrocene is present in the reaction
medium, it acts as an electron donor during oxidation of phe-
nolic acids. Mediator is oxidised by the working electrode. As
a result, current is decreased and detected using the electrode
system (Fig. 2).
In this study, alternative systems were developed for faster
and cheaper determination of phenolic acids in real samples
of human plasma by bioanalytical methods including Trametes
versicolor laccase (TvL) and two different transducers: a com-
Fig. 2. Principle of ferrocene-mediated laccase biosensor.
2. Materials and methods
All chemicals were commercially available and of reagent
grade. Three-hundred bloom calf skin gelatin, glutaralde-
hyde (GA), ferulic acid, caffeic acid, syringic acid and 2,2-
azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS)
were obtained from Sigma Chem. Co. (St. Louis, MO,
Plasma samples were obtained from a local hospital and
WTW InoLab Oxi Level 2 model dissolved oxygenmeter
based on amperometric mode and WTW Cellox 325 Dis-
Chronoamperometric measurements were carried out with the
Radiometer (Voltalab PGP 201) electrochemical measurement
system (France). Ag/AgCl and Pt were used as reference and
counter electrodes, respectively.
2.3. Electrode preparation
Ferrocene-modified screen-printed electrodes were home
made prepared on PVC substrates by using a HT10 Machine
(Fleishle, Germany). Pastes for printing, working electrodes
were prepared by using a commercially available graphite paste
(GWENT Electronics Materials Inc.®). Printed electrodes were
fabricated by depositing several layers of pastes on a PVC
substrate. 5.6% Ferrocene (w/w) was added to graphite paste,
the graphite working electrode, auxiliary electrode, conduct-
ing paths and pads were deposited directly on the PVC sheets
using graphite pastes (GWENT Electronics Materials Inc.®).
Then, an insulator paste (GWENT Electronics Materials Inc.®)
was deposited. Finally, an Ag/AgCl paste (GWENT Electron-
ics Materials Inc.®) was placed over the conducting paths
D. Odaci et al. / Talanta 71 (2007) 312–317
Fig. 3. SPG electrodes: from left to right the concentric lay-out of sequentially
printed layers: (a) ferrocene/graphite auxiliary and working electrodes, conduc-
tive paths and pads; (b) insulator; (c) Ag/AgCl reference electrode.
2.4. Culture medium of microorganism and laccase
gus T. versicolor (ATCC 11 235). T. versicolor was maintained
at 4◦C on 2% malt agar and grown in 100ml malt extract broth
limited medium consisting of 10g glucose, 1g NH4H2PO4,
0.05g MgSO4·7H2O, 0.01g CaCl2and 0.025g yeast extract,
per litre. The cultures of T. versicolor were incubated at 26◦C
on a rotary shaker at 175rpm. After 72h cultivation, growing
medium was used as a source of enzyme. Laccase production
was assessed by measurement of enzyme oxidation of 2,2-
azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) at 427nm
(ε=3.6×104cm−1M−1) . The reaction mixture contained
300?L of extracellular fluid, 300?L of 1mM ABTS and 0.1M
as the amount of enzyme that oxidises 1?mol ABTS in 1min.
Final activity for TvL was 350Uml−1.
2.5. Preparation of biosensors
Both commercial oxygen electrode and ferrocene-modified
screen-printed electrode were used for preparation of biosen-
sors. Gelatin (10mg) and 4U of laccase in 250?l of phosphate
buffer (pH 7.5, 50mM) were mixed at 38◦C for few minutes.
Then, the solution spread over probe surface and allowed to dry
at 4◦C for 45min. Moreover, for screen-printed electrodes the
procedure was the same as reported above, with the exception
of the deposition of 2.5?l of mixed solution on the surface of
ferrocene-modified graphite working electrode. Four units lac-
case was used for both biosensors. Finally, they were immersed
in 2.5% glutaraldehyde in 50mM phosphate buffer (pH 7.5)
for 5min . Thus, Type I biosensor including dissolved oxy-
gen probe and Type II biosensor including ferrocene-modified
screen-printed electrodes were prepared for detection of pheno-
detected with Type I biosensor to determine the concentration
of phenolic acids. All the measurements were done at 35◦C
under continuous and constant stirring and varying substrate
reaction cell. Enzymatic reaction was completed in 10min. The
tion of the measurement, the electrode was rinsed with distilled
water and allowed to equilibrate before the following measure-
ment. Fifty millimolars of acetate buffer, pH 4.5, was used as
working buffer for laccase biosensor.
its oxidation took place in the bioactive layer and was sensed as
a change in the current intensity with a potentiostat at 0.28V
versus Ag/AgCl pseudo-reference electrode.
3. Results and discussion
3.1. Optimisation of assay conditions
mercial oxygen electrode, while all the remaining experiments
have been done with both biosensors.
3.1.1. Effect of pH
Fig. 4(a) and (b) show the results obtained from pH optimi-
sation studies of the biosensors. The optimal pH value of 4.5
of both systems was obtained for caffeic acid, ferulic acid and
syringic acid. A decrease was observed for lower and higher pH
where the variation of laccase activitiy from different enzyme
sources with pH was tested by using phenol as a substrate.
3.1.2. Effect of temperature
Electrode response was measured at different temperatures
ranging from 25◦C to 40◦C with Type I biosensor. As shown in
Fig. 5, the maximum response was obtained at 35◦C, which is
also known as optimum temperature value for laccase enzymes
temperature was used for all measurements.
3.1.3. Thermal stability
The thermal stability experiments were performed at work-
ing conditions (acetate buffer, pH 4.5, 50mM and 35◦C) for
Type I biosensor. Our data showed no decrease in biosensor
activity of the biosensor was observed up to 8h. In this period,
approximately 28 measurements have been made. It could be
D. Odaci et al. / Talanta 71 (2007) 312–317
Fig. 4. Effect of pH on Type I (a) and Type II; (b) biosensor responses (pH
3.0–5.5, 50mM acetate buffer, 35◦C).
3.2. Effect of applied potential
Hydrodynamic voltammograms of Type II biosensors based
on graphite-ferrocene inks (Fig. 6) were previously performed
in our laboratories with several immobilisation procedures and
enzymes (peroxidase, tyrosinase, laccase), which have phenols
as substrates. Tyrosinase  and peroxidase were immobilised
whereas TvL was immobilised as described above. Similar
behaviour of the three biosensors (peroxidase voltammogram
as a consequence of experimental conditions and immobilisa-
tion procedures used. The optimal potential for Type II biosen-
sor (+280mV versus Ag/AgCl pseudo-reference electrode) was
chosen according to Fig. 6.
Fig. 6. Hydrodynamic voltammograms obtained with ferrocene-graphite elec-
trodes coupled with cross-linked tyrosinase or TvL. Response to 1mM phenol
(?) TvL in a gelatin matrix (acetate buffer; 50mM, pH 4.5, 35◦C).
3.3. Analytical characteristics
Linearity was obtained in a concentration range 0.1–1.0?M
caffeic acid, 0.05–0.2?M ferulic acid, 2.0–14.0?M syringic
acid for laccase immobilised on commercial oxygen elec-
trode and 2.0–30.0?M caffeic acid, 2.0–10.0?M ferulic
acid, 4.0–30.0?M syringic acid for laccase immobilised on
ferrocene-modified screen-printed electrodes (Table 1). At
higher concentrations, calibration curves showed a deviation
are shown in Table 1 as coefficient of variation (CV).
On the other hand, ascorbic acid, uric acid and paraceta-
mol which could be catalytically oxidised by ferricinum ion
were tested at different concentrations. No signal was detected
with Type II biosensor at +280mV at concentrations lower than
50?M ascorbic acid, 100?M uric acid and 25?M paraceta-
mol. These concentrations are relatively higher than detection
ranges for phenolic acids tested in this paper. At higher concen-
for ascorbic acid (50?M), uric acid (100?M) and paracetamol
3.4. Phenolic acid determination of human plasma
Proposed biosensors were analytically evaluated with real
samples of human plasma. TvL is specific for a class of com-
pounds, not for a single compound and this characteristic has
been exploited to obtain an analytical device for a wide spec-
trum of phenolic compounds. In addition, TvL shows different
specificity for its substrates. Accuracy is then affected by the
different specificity of the enzyme for all substrates. For this
ferred with respect to the comparison with a reference method.
For this purpose, denaturated plasma sample was prepared as
described in material and method and used as stock solution.
Plasma samples were added to reaction cell after equilibration
and changes in the responses were recorded for both types of
biosensors. Then, plasma samples with spiked amount of sev-
were calculated from the calibration curves. All results includ-
ing recovery coefficients (Table 2) are analytically acceptable.
D. Odaci et al. / Talanta 71 (2007) 312–317
Analytical features of the proposed laccase biosensors
Type I biosensorType II biosensor
Slope of the calibration lineb
Correlation coefficient (R2)
Linearity range (?M)
bSlope is given in mgL−1?M−1for Type I biosensor and in ?Acm−2?M−1for Type II biosensor.
cLOD (limit of detection) and LOQ (limit of quantitation) have been calculated with the graphical method reported by Meier and Zund .
dRepeability measurements and standard deviation (σ) calculation were performed with n=5 replicates of 0.75?M caffeic acid, 0.1?M ferulic acid, 10.0?M
syringic acid with Type I biosensor and 4.0?M caffeic acid, 6.0?M ferulic acid, 10.0?M syringic acid with Type II biosensor.
eCoefficient of variation.
Application of biosensors to human plasma
Amount of phenolic acid
in plasma (?M)a(C0)
Added phenolic acid
Detected total phenolic
acid (?M) (Ctot)
Type I biosensor
0.58 ± 0.03
0.78 ± 0.04
0.13 ± 0.01
0.18 ± 0.01
2.1 ± 0.6
10.1 ± 0.3
Type II biosensor
2.2 ± 0.5
4.8 ± 0.8
2.2 ± 0.4
5.0 ± 0.3
6.1 ± 0.5
11.6 ± 0.3
All results are given as value±S.D., n=5.
aAdded phenolic acid concentration were calculated after addition to the blank plasma sample using the standard curves obtained for each substrate.
The nature of sample does not affect the measurements. More-
over, interferences of some compounds were already mentioned
in Section 3.3. Apart from these compounds, electrochemical
response of each phenolic acid on naked screen-printed elec-
was obtained in each case. All data showed that in the detection
ranges both types of biosensor could be used as an analytical
system selective for the class of phenolic compound.
mination of phenolic acids. Optimal working conditions were
modified oxygen electrode. Under these conditions, calibration
analytical parameters, reproducibility studies, including sam-
ple application, were done for both laccase modified electrodes.
Laccase based biosensors are simple, rapid to prepare, reliable
fast and of low cost. Furthermore, they offer a good method
for screening phenolic acids in complex matrices like human
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