Available online at www.sciencedirect.com
Biosensors and Bioelectronics 23 (2008) 1733–1737
CYP450 biosensors based on gold chips for antiepileptic
M.A. Alonso-Lomilloa,∗, J. Gonzalo-Ruizb,1, O. Dom´ ınguez-Renedoa,2,
F.J. Mu˜ nozb,1, M.J. Arcos-Mart´ ıneza,2
aDepartment of Analytical Chemistry, Faculty of Sciences, University of Burgos, Burgos, Spain
bMicroelectronics Institute of Barcelona (IMB-CSIC), Bellaterra-Barcelona, Spain
Received 20 September 2007; received in revised form 17 December 2007; accepted 29 January 2008
Available online 8 February 2008
Three-electrode configuration chips containing a Pt, Au and a screen-printed Ag/AgCl as counter, working and reference electrode, respectively,
have been developed. Selective determination of Phenobarbital (PB) has been carried out by Cytochrome P450 2B4 (CYP450) immobilization into
a polypyrrole matrix onto the gold working electrode. Chronoamperometric experiments show a PB diffusion coefficient of 2.42×10−6cm2s−1,
a reproducibility and repeatability in terms of residual standard deviation (RSD) of 13% and 5.51%, respectively, and a limit of detection (LOD)
of 0.289?moldm−3(α=β=0.05) for the developed CYP450-biosensor chip. Its performance has been showed by the determination of PB in
pharmaceutical drugs. HPLC has been used as reference technique.
© 2008 Elsevier B.V. All rights reserved.
Keywords: Biosensor; Microelectronic techniques; Screen-printed Ag/AgCl reference electrode; Phenobarbital; Cytochrome P450 2B4
from millions people worldwide. Epilepsy is usually controlled
and Feuerstein, 2007) have been developed.
Anticonvulsant drugs of the first generation – phenobarbi-
tal (PB), primidone (PRD), phenytoin (PHT), carbamazepine
(CBZ) and valproic acid (VPA) – have an increased potential
for interactions and side effects due to enzyme induction and/or
inhibition. Long-term use can give rise to changes in bone and
sexual dysfunction (Stefan and Feuerstein, 2007). Therapeutic
drug monitoring can increase the clinical efficacy while mini-
mizing adverse effects (Zhang et al., 1999).
In 1949, a colorimetric method for the determination of
PB was described (Jones and Howland, 1949). Since then,
hundreds of PB determinations have been described based
∗Corresponding author. Tel.: +34 947258818; fax: +34 947258831.
E-mail address: firstname.lastname@example.org (M.A. Alonso-Lomillo).
1Tel.: +34 935947700; fax: +34 935801496.
2Tel.: +34 947258818; fax: +34 947258831.
on spectrophotometric (Boeris et al., 2000), chromatographic
(Cavazos et al., 2005; Vermeij and Edelbroek, 2007), phos-
phorimetric (Wineford and Tin, 1965), liquid–liquid extraction
and immunoassay methods (Sayo et al., 1988). In spite of the
high sensitivity, simplicity and inherent miniaturization of elec-
trical assays, only a few electrochemical determinations have
been reported (Bordes et al., 1999; Ni et al., 2004; Romer et
al., 1977; Temizer and Solak, 1986; Zhang et al., 1999). More-
over, the combination of an electrochemical transducer and a
biological element, which confers high specificity to the sensor,
has only been shown in the case of electrochemical immunoas-
says. Due to their specificity, speed, portability and low cost,
biosensors offer exciting opportunities for numerous decentral-
has been developed in this research work for the determination
of PB in different kind of samples, such as synthetic samples
(model solution) and pharmaceutical preparations.
CYP450 is an important family of heme-containing hydrox-
ylases, found in most organisms from bacteria to human beings
(Metzler, 2001). PB is one of the preferred CYP450 2B sub-
strates. The general characteristic of this enzyme substrates
0956-5663/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
M.A. Alonso-Lomillo et al. / Biosensors and Bioelectronics 23 (2008) 1733–1737
include non-planar molecular geometries couple with a rela-
tively high lipophilicity, together with the presence of one or
two key hydrogen bond-forming groups and, usually, at least
one aromatic ring adjacent to a tetrahedral carbon often impart-
ing a V-shaped conformation to the structure (Lewis, 2001). In
essence, the CYP450-mediated reaction involves the combina-
tion of oxygen with the organic substrate (RH) to produce a
molecule of water and a mono-oxygenated metabolite (ROH)
(Metzler, 2001; Shumyantseva et al., 2004).
CYP450 2B4 can be immobilized onto the working elec-
trode surface by electropolymerization. Thus, native structure
and appropriate orientation can be achieved, increasing elec-
tron transfer between the enzyme and the electrode (Bistolas
et al., 2005). Here, CYP450 2B4 has been immobilized onto
a gold working electrode by pyrrole (py) electropolymeriza-
tion. Polypyrrole (PPy) is one of the most studied conducting
polymers because of its easy preparation, high conductivity and
relative stability (Sabah et al., 2006).
As only those biomolecules present in the vicinity of the
electrode surface are incorporated in the growing polymer
(Cosnier et al., 1998), high concentrations of biomolecules in
the aqueous electrolyte during the electropolymerization pro-
cess are required. Consumption of large quantities of biological
reagents can be avoided using electrochemical cells based on
microelectronic configurations. Therefore, miniaturized amper-
ometric silicon-based transducers (3mm×3mm), containing
of photolithographic techniques (Alonso-Lomillo et al., 2005).
been printed at wafer level by screen-printing techniques.
Experimental variables in the chronoamperometric determi-
nation of PB have been optimized by means of the experimental
design methodology. Reproducibility, repeatability and limit of
been applied to the PB determination in pharmaceutical drugs.
2.1. Reagents, equipment and software
In the fabrication of the screen-printed reference electrode
at wafer level, Ag/AgCl ink (Electrodag 6037) from Acheson
Colloids Company (Port Huron, USA) was used.
CYP450 2B4 (C9095-15Y, USBiological, Swamscott, Mas-
sachusetts, USA) was used as received.
Solutions of 0.05moldm−3pyrrole (Sigma, Steinheim, Ger-
many), 0.1moldm−3LiClO4(Panreac, Barcelona, Spain) were
prepared by dissolving the adequate amount of each one in
used as supporting electrolyte.
For the chromatographic analysis, acetonitrile–water (25:75,
v/v) adjusted to pH 2.5 with H3PO4was used as mobile phase.
Stock solutions were prepared by dissolving the appropriate
amount of PB (Sigma, Steinheim, Germany) in ethanol–water
(10:90, v/v) and in mobile phase for the electrochemical and
chromatographic measurements, respectively.
Voltammetric and amperometric measurements were carried
out in bulk solution using a ?Autolab Type II Potentiostat (Eco
Chemie, BV, The Netherlands).
HPLC analysis was performed on a chromatographic system
a Knauer injector and a 254nm fixed wavelength detector K-
200 (Knauer, Berlin, Germany). The HPLC column (VERTEX
column, Eurosphere 100 C18, 5?m, 125mm×4mm) was also
bought from Knauer (Berlin, Germany).
Amperograms and voltamperograms were acquired and pro-
cessed by using the General Purpose Electrochemical System
Software. A EuroChrom 2000 software was used to collect,
integrate and analyse the chromatographic data.
1994–2001) was used for experimental design and data
analysis, PROGRESS for the robust regressions (Rousseeuw
and Leroy, 1989) and DETARCHI for estimation of the
detection limit (Blanco et al., 1994; ISO11843-2, 2000).
2.2. Electrode preparation
Three-electrode configuration chips (3.0mm×3.0mm)
were fabricated over a silicon wafer by photolithographic and
screen-printing techniques (Fig. 1).
Thermal oxide layer (800nm thick) was grown as insulating
substrate on a 4 in, diameter silicon wafer. This was followed
by a metallization step, carried out by electron gun system. A
titanium layer acting as adhesion promoter (20nm) was fol-
lowed by a platinum layer (150nm). Subsequent patterning
was done by lift-off procedure in order to form the platinum
electrodes. Following this step, Ti (20nm), Ni (20nm) and Au
(20nm) were deposited by physical vapour deposition (PVD)
and patterned by wet etching to define the gold working elec-
Fig. 1. Microscopic image of a three-electrodes configuration chip developed
by photolithographic and screen-printing techniques.
M.A. Alonso-Lomillo et al. / Biosensors and Bioelectronics 23 (2008) 1733–1737
a passivation layer was deposited onto the wafer for electrical
interference prevention and chemical protection. Silicon oxyni-
tride layer (1?m) was grown by plasma-enhanced chemical
vapour deposition (PECVD-SiOxNx). This layer was patterned
and those areas acting as working (0.5mm2), counter electrode
(1.36mm2) and a reference electrode (0.35mm2) were opened
trode was fabricated by conventional screen-printing technique
(Desmond et al., 1997), over the electrode defined as reference
amperometric transducers at wafer level. Then, a drying cycle
(80◦/30min+120◦/5min) was applied.
After the fabrication at wafer level, the chips were separated
and surface mounted on test printed circuit boards (PCB), using
an UV-curable polymer as encapsulant.
Gold working electrodes were first treated by successive
cycling in buffer solution between −1.2V and −2.2V vs.
Ag/AgCl, 3moldm−3KCl (10 cycles; scan rate, 100mV/s) to
clean and activate the electrode surface. This procedure was
performed until reproducible phosphate voltammograms were
recorded between −0.5V and 0.3V vs. Ag/AgCl, 3moldm−3
KCl. This pretreatment was employed before each electropoly-
2.2.1. CYP450 2B4 immobilization
was first deposited by scanning the potential between 0V and
+0.9V vs. Ag/AgCl 3moldm−3at a scan rate of 10mVs−1,
for one complete cycle, at room temperature (Alonso-Lomillo
et al., 2003, 2005). The screen-printed reference electrode was
used, which allows a small volume of solution (1ml) to be
used. The oxidized form of the polymer is positively charged
and requires the incorporation of doping anions, LiClO4,
in order to maintain its electroneutrality (Gros and Comtat,
2004). The association between polymer cations and dopant
anions depends on the electrochemical conditions applied dur-
ing the process of polymerization. So, in addition to the proper
distribution of positive charge for anion recognition func-
tionality, the polymers have to carry pores of suitable size,
which form the host cavities for dopant anions. These steric
requirements can restrict possible interference from other ions.
Therefore, the sensors based on polymers, which have such
additional recognition principles could also have better selec-
tivity in comparison to other types of sensors (Sabah et al.,
The PPy-CYP450 2B4 film was subsequently grown onto
the gold working electrode. The electrode was immersed in a
solution containing 0.1moldm−3LiClO4, 0.05moldm−3pyr-
role, and 0.133% (v/v) of the CYP450 2B4 solution at room
temperature. The potential domain was scanned twice between
0V and +0.9V vs. Ag/AgCl 3moldm−3at a scan rate of
After each measurement, the biosensor was regenerated by
immersing it in an unstirred blank buffer solution for 30min
at 4◦C, in order to wash away the analyte from the polymer
3. Results and discussion
3.1. Sensor characterization
First, the stability of the developed reference electrode was
tested. Its potential stability was measured in a 3moldm−3
KCl solution vs. a conventional reference electrode Ag/AgCl
3moldm−3. A medium value of potential of 62.0±0.89mV
(n=3, α=0.05), with a drift lower than 0.23mVh−1, was
recorded for at least 10h, with an elapse time of 2h due to
the electrode hydration.
Moreover, the long-term stability was checked by measuring
measurement was constant for 20 days.
tial generated by the Ag/AgCl electrode is directly proportional
to the chloride ion activity and, at 25◦C,
E = E0− 59.16 log aCl−.
Several calibration curves were performed by ranging the
chloride concentration from 0.001moldm−3to 0.1moldm−3.
A slope of 56.8±1.5mV was obtained, which indicates a
Nernstian behaviour for the developed screen-printed reference
The behaviour of the reference electrode was studied when
the chloride concentration drastically changes (3moldm−3
and 0.1moldm−3). An equilibrium potential was immediately
reached, keeping a stable value of potential while chloride con-
centration is not changed.
Finally, the performance of the reference electrode was also
checked by recording the typical cycle voltammograms of a
5mmoldm−3ferricyanide in 0.1moldm−3phosphate solution
the developed electrode and a conventional one (3moldm−3
as inner solution), obtaining good agreement between them
Fig. 2. Cyclic voltammograms of a 5mmoldm−3ferricyanide solution using
screen-printed Ag/AgCl (solid line) and a commercial Ag/AgCl 3moldm−3
(dash line) reference electrodes. Scan rate: 100mVs−1.
M.A. Alonso-Lomillo et al. / Biosensors and Bioelectronics 23 (2008) 1733–1737
ent concentration of PB, a–j: 0.32, 0.63, 0.91, 1.18, 1.43, 1.67, 1.89, 2.11, 2.31,
2.50?moldm−3. pH 9 and operating potential (Eap)=0.45V.
3.2. Determination of the diffusion coefficient of PB
The diffusion coefficient of PB in solution can be estimated
by using chronoamperometric experiments (Yu et al., 2007) and
I = nFAD1/2cπ−1/2t−1/2
where D and c are the diffusion coefficient (cm2s−1) and bulk
concentration (molcm−3), respectively, and A was the electrode
Fig. 3 shows the chronoamperograms at various PB concen-
trations for a CYP450 2B4-PPy gold biosensor chip. The plot of
the current controlled by the diffusion of PB from the bulk solu-
slopes of the resulting straight lines were then plotted vs. the PB
concentration, from whose slope (2.66) the diffusion coefficient
D could be calculated. According to the well-accepted general
mechanism of the CYP450, two electrons are transferred in the
reactions (Lewis, 2001; Shumyantseva et al., 2004). Thus, the
diffusion coefficient was found to be 2.42×10−6cm2s−1.
3.3. Chronoamperometric determination of PB at CYP450
2B4-PPy gold biosensor chip
stitution of the biological electron delivery and transport system
by the electrode (Shumyantseva et al., 2004). In order to get the
maximum current response for PB, the operating potential (Eap)
in chronoamperometry and the pH of the solution were inves-
tigated. A 22central composite design was carried out for the
optimization of these parameters, taking the intensity registered
at 50s for a 3.22×10−5moldm−3solution of PB as response
(Alonso-Lomillo et al., 2003).
From the optimisation process based on the experimental
design methodology, the following optimum values for the
experimental variables in the PB determination were taken
gradually and has been attributed to a decrease in conductivity
of the film due to oxidation reactions (Sabah et al., 2006).
Fig. 4 shows the chronoamperogram of the modified
electrode during the successive addition of 100?l of a
10−5moldm−3PB solution, registered under the optimum con-
ditions. Measurements were taken under stirring conditions.
Control experiments were done in which no CYP450 was
deposited. A poor chronoamperometric response for PB was
obtained in comparison to the developed biosensor.
The chronoamperograms recorded at 1.43?moldm−3of a
ity and repeatability. Residual standard deviation (RSD) values
of 13.68% and 5.51% (n=3, α=0.05) were obtained, respec-
The limit of detection (LOD) was also assessed. Calibration
curves in the concentration range 0.32–2.50?moldm−3of PB
The parameters of these calibrations were optimally evaluated.
In order to avoid incorrect adjustments due to the existence of
anomalous points, least median squares regression (LMS) was
used (Massart et al., 1986; Rousseeuw and Leroy, 1989). Then,
the detection limit was calculated using the DETARCHI pro-
gram, with evaluation of the probability of false positive (α) and
negative (β) (Alonso-Lomillo et al., 2003; Blanco et al., 1994;
Antiepileptic drugs such as primidone, ethosuximide and
phenytoin did not hinder the analysis of PB, since at a con-
centration of 1.64×10−5moldm−3these compounds gave no
response under the optimised conditions.
cessive addition of 100?l of a 10−5moldm−3PB solution, registered under the
M.A. Alonso-Lomillo et al. / Biosensors and Bioelectronics 23 (2008) 1733–1737 Download full-text
3.4. Determination of PB in pharmaceutical drugs
determination of PB in LUMINALETAS®by Kern Pharma. A
tablet was dissolved into ethanol-water (10:90, v/v), sonicated
until complete dissolution, centrifuged and finally, filtered.
Next, the PB concentration was evaluated by comparison
with a standard addition of identical volume (50?l) of a
10−3moldm−3PB solution to a sample of the dissolved drug.
This value was checked by HPLC determination. A calibra-
range 0.004–0.0032mgmL−1for PB. The PB peak area found
in the LUMINALETAS®solution was [0.564±0.028] (n=5,
α=0.05), which corresponds to 4.049mmoldm−3of a PB in
the stock solution.
Since PB is one of the preferred substrates of CYP450, this
enzyme has been used as the biological component in the devel-
opment of biosensors for the selective determination of this
antiepileptic drug. In order to use a small cell volume and,
therefore, small amount of reagents, three-electrode configu-
rations chips have been fabricated by photolithographic and
screen-printing techniques. Screen-printed Ag/AgCl reference
electrodes have been characterized. The viability of these elec-
trodes for further applications has been showed. Thus, CYP450
was immobilized by Py electropolymerization onto the gold
Chronoamperometric experiments enabled determination of
ferent kind of samples. Once experimental variables have been
ducibility, repeatability and LOD have been calculated. Finally,
the performance of the biosensor has been checked by apply-
ing it to the determination of PB in pharmaceutical drugs. PB
concentration has been analyzed in LUMINALETAS®by the
standard addition method. HPLC, which has been used as ref-
erence technique, showed good agreement with the proposed
Authors would like to acknowledge funding via the Spanish
Ministry of Education and Science (MAT2005-01767) and Dr.
J.M. Trejo from the Hospital General Yag´ ue in Burgos for his
y Cajal fellowship from the Spanish Ministry of Education and
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