|Título/s:||Screen-printed electrochemical biosensors based on magnetic core-shell nanoparticles|
|Autor/es:||Longinotti, G.; Lloret, P.; Ybarra, G.; Moina, C.; Hermida, L.; Milano, O.; Roberti, M.; Malatto, L.; Fraigi, L.|
|Institución:||INTI-Procesos industriales. Buenos Aires, AR |
INTI-Química. Buenos Aires, AR
INTI-Electrónica e Informática. Buenos Aires, AR
|Palabras clave:||Biosensores; Sensores electroquímicos; Películas delgadas; Serigrafía; Nanopartículas; Enzimas|
| Ver+/- |
Screen-printed electrochemical biosensors based on magnetic
Longinotti, G.; Lloret, P.; Ybarra, G.; Moina, C.
Centro de Procesos Superficiales, Instituto Nacional de Tecnología Industrial (INTI), Buenos Aires, Argentina.
Centro de Química, Instituto Nacional de Tecnología Industrial (INTI), Buenos Aires, Argentina.
Milano, O.; Roberti, M.; Malatto, L; Fraigi, L.
Centro de Electrónica e Informática, Instituto Nacional de Tecnología Industrial (INTI), Buenos Aires,
Abstract. In this work, the design, manufacture and characterization of biosensors based on thick film (screen printing)
technology is presented for the electrochemical determination of the catalytic activity of enzymes immobilized onto
superparamagnetic iron oxide nanoparticles (SPION).
Keywords: Biosensors, Photolithography; Screen Printing; SPION; Enzymes.
Enzymatic electrodes are a type of biosensors formed
by an electrode on whose surface a redox enzyme has been
immobilized . Enzymes catalyze reactions producing
redox substances which can be electrochemically detected
at the electrode; thus, the resulting current is a measure of
the reaction rate. Usually, the analytes in enzymatic
electrodes are the substrates of the enzymes (i.e. the
substance whose reactions are catalyzed by the enzyme); in
this case, a higher current at the electrode is a consequence
of a higher concentration of the analyte. Enzyme inhibitors
(i.e. substances which inhibit the catalytic activity of the
enzymes) can also be regarded as analytes. For instance,
acetylcholinesterase (AChE) is an enzyme which
participates in neurotransmission process and is inhibited
by organophosphorus and carbamate insecticides. In these
cases, a lower current at the electrode is a consequence of a
higher concentration of the analyte.
The immobilization of enzymes onto nanoparticles and
the subsequent attachment of the nanoparticles onto an
electrode is an attractive alternative in this context;
especially in the case of magnetic nanoparticles which can
be attracted to and removed from the electrode by the
action of magnetic fields. Such enzyme modified magnetic
nanoparticles may be used in a pre-concentration step for
the determination of inhibitory substances. Another
attractive aspect is the possibility of using a single working
electrode for the determination of multiple, sequential
analytes simply by changing the enzymes attached to the
In this work, the feasibility of the electrochemical
determination of the catalytic activity of horseradish
peroxidase (HRP) immobilized onto chitosan modified
superparamagnetic iron oxide nanoparticles (SPION)
employing a miniature electrochemical cell is presented.
II. EXPERIMENTAL DETAILS
Electrodes design and manufacture
The sensor design, made up of three electrodes, was
developed on the basis of a previous one carried on Au
electrodes deposited on Si and glass substrates. Thick film
electrodes were printed by conventional screen printing
technology. Commercial Au organometallic paste (ESL
D8083) and 96 % α-Al2O3 substrates were employed. The
three-electrode layout was transferred by means of
photolithography to a stainless steel mesh (400 wires per
inch) with a photosensitive film (Ulano CDF-2).
HRP was immobilized on screen printed Au electrodes
employing cysteine and glutaraldehyde as molecular
linkers between the gold electrode and the enzyme.
Nanoparticles synthesis and enzyme immobilization
Chitosan coated SPION were obtained by a co-
precipitation method . Briefly, a solution containing 0.5
% chitosan and stoichiometric quantities of Fe2+ and Fe3+
was mixed with NH4OH solution under stirring. After 30
min, the precipitate was magnetically collected, washed,
re-dispersed in water, and stored. The quantity of chitosan
in the shell, determined by the ninhydrin method , was
12 mg per 100 mg of SPION.
Chitosan coated SPION were immersed in a 7 %
glutaraldehyde solution overnight, washed with deionized
water, immersed in 0.1 M phosphate buffer of pH 7
containing a 2.49 U µl-1 HRP during 2 hours, and finally
washed six times with 100 µl of deionized water.
The catalytic activity of the HRP on the SPION was
confirmed with a colorimetric technique. When 4-
aminoantipyrine is employed as an electron donor by HRP
during the oxidation of H2O2, it generates a colored
substance (quinoneimine) in the presence of phenol. 50 µl
of an aqueous dispersion of SPION was added to 3 ml of
0.1 M phosphate buffer of pH 7 containing 0.4 mM 4-
aminoantipyrine, 0.63 mM phenol 0.63 and 0.8 mM H2O2;
the absorbance at 500 mm was recorded.
0 10 20 30
t / min
Fig. 1. Time evolution of the absorbance at 500 nm of SPION-HRP.
Current-potential curves were obtained at a scan rate of 20
mV s-1 in a 0.1 M phosphate buffer of pH 7 + 0.1 M KCl. 4
mM hydroquinone was employed as a redox mediator and
the H2O2 concentration was increased from 0.2 to 1.2 mM.
AgAgCl was employed as a reference electrode. SPION
were collected with the aid of a magnet and concentrated
on the surface of the screen printed gold electrode.
The time evolution of the absorbance of the SPION-
HRP is shown in Fig. 1. The catalytic activity of the
enzyme is retained after the immobilization onto the
nanoparticles. Fig. 2 shows the current-potential curves
obtained for the electrodes with enzymes immobilized onto
Au electrodes (Fig. 2a) and enzymes immobilized onto
SPION (Fig. 2b). The resulting calibration curves are
presented in Fig. 3. The sensitivity is quite similar for both
-0.2 -0.1 0.0 0.1
E / V
-0.30 -0.25 -0.20 -0.15
E / V
Fig. 2. Current-potential curves obtained with solution of y mM H2O2 (y = 0,
0.6, 1.0). a) Enzymes immobilized onto Au electrodes; b) Enzymes
immobilized onto SPION.
0.0 0.5 1.0 1.5 2.0
C / mM
0.0 0.5 1.0
C / mM
Fig. 3. Current (at -0.190 V) vs. H2O2 concentration curves obtained for: a)
Enzymes immobilized onto Au electrodes; b) Enzymes immobilized onto
The electrochemical response of the enzymes
immobilized onto SPION was comparable to that obtained
for enzymatic electrodes. Measurements in a conventional
electrochemical macro-cell were performed in order to
asses the influence of the scaling-down in the response of
These results open the possibility for the design and
fabrication of biosensors for monitoring the quality of
waters employing enzymes immobilized onto SPION as
 Solé S., Merkoci A., Alegret S.; Crit. Rev. Anal. Chem
33 (2) (2003) 89.
 H. Honda et al; J. Fermentation Bioengineering 86 (2)
 Shi-Wen Sun et al; J. Food Comp. Anal. 19 (2006)