|Título/s:||Diagnosis of foot-and-mouth disease by electrochemical enzyme-linked immunoassay|
|Autor/es:||Longinotti, Gloria; Ybarra, Gabriel; Lloret, Paulina; Moina, Carlos; Ciochinni, Andrés; Rey Serantes, Diego; Malatto, Laura; Roberti, Mariano; Tropea, Salvador; Fraigi, Liliana|
|Institución:||INTI-Procesos Superficiales. Buenos Aires, AR |
INTI-Electrónica e Informática. Buenos Aires, AR
Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. UNSAM. Buenos Aires, AR
|Palabras clave:||Inmunosensores; Inmunoensayos; Diagnóstico; Fiebre aftosa; Detección; Detectores; Métodos electroquímicos; Enzimas|
| Ver+/- |
Abstract—The development of an inmunosensor for the
point-of-care detection of the foot-and-mouth cattle disease is
presented. The detector is based on an ELISA method with
electrochemical detection. A non-structural protein, 3ABC, is
used to selectively detect antibodies is used to selectively detect
anti-3ABC antibodies produced after infection. The biological
test is performed onto a screen printed electrodes. A dedicated
small, portable potentiostat is employed for the control of the
sensors, as well as data acquisition, processing, and storage.
Biosensors are compact analytical devices which employ a
biological element in order to detect a specific substance (i.e.
the analyte). After the analyte has been detected by the
biological recognition element, a signal of physical or
chemical nature is produced which then is converted into an
electrical signal by means of a transducer. In the case of
electrochemical biosensors, a chemical signal generated by
the interaction between the biological recognition element and
the analyte is converted into an electrical current via an
electrochemical reaction at an electrode surface . In this
paper, we present the method and instrumentation for an
electrochemical enzyme-linked immunoassay for the
diagnosis of the foot-and-mouth disease. This presentation
covers all aspects of the development of the biosensor: the
production of gold electrodes by thick film technology and
electrochemical cells with a numeric control device, the
chemical modification of the gold electrodes with the
biological recognition elements, which are antigens of proteic
nature, the electrochemical transducer via the action of a
redox mediator (a chemical substance which acts as the
electrical connection between the enzymes and the electrode),
and finally the electronic instrumentation required to control
the electrochemical system and process the resulting signal
[2,3]. Results obtained for the diagnosis of the foot-and-mouth
disease are presented.
II. EXPERIMENTAL DETAILS
A. Electrodes design and manufacture
A three-electrode cell was designed and manufactured.
Ring-disk geometry was defined for the working and counter
electrodes. Disks with a diameter of 1000 µm were employed
*Corresponding author: firstname.lastname@example.org. This work was supported by
the Instituto Nacional de Tecnología Industrial and the Instituto de
as working electrodes. Counter electrodes were designed to
have ten times as area as the working electrodes. The inner
and outer diameters of the rings were 1600 µm and 3600 µm.
The resulting gap between electrodes was 300 µm. Trace
width was 300 µm and pad size was 2 mm × 2 mm for both
electrodes. A 3 × two-electrode array was designed, so they
could be evaluated together or individually (Fig. 1).
Fig. 1. a) Photograph of a thick film two-electrode configuration showing
active circular area with 1000 µm diameter for the working electrode. b)
Three pairs of electrodes as used in the electrochemical cells.
The two thick film electrodes were printed onto α-Al2O3
substrates by conventional screen printing technology. A wire
of 1 mm diameter of AgAgCl was employed as a reference
electrode. Commercial Au paste (Heraeus D5789) and 96 %
α-Al2O3 substrates were employed. Electrode layout was
transferred by means of photolithography to a stainless steel
mesh (400 wires per inch) with a negative photosensitive film
(Ulano CDF-2). Au ink printing was performed by an EKRA
Microtronic-II printer, dried at 125ºC during 15 min and
finally fired at 580°C.
Fig. 2. Acrylic electrochemical cell with thick film gold electrodes.
Diagnosis of Foot-and-Mouth Disease by Electrochemical
Gloria Longinotti,1 Gabriel Ybarra,1 Paulina Lloret,1 Carlos Moina,1,*Andrés Ciochinni,2 Diego Rey
Serantes,2 Laura Malatto,1 Mariano Roberti,1 Salvador Tropea1 and Liliana Fraigi1
1Instituto Nacional de Tecnología Industrial, Argentina.
2Instituto de Investigaciones Biotecnológicas, UNSAM, Argentina.
32nd Annual International Conference of the IEEE EMBS
Buenos Aires, Argentina, August 31 - September 4, 2010
978-1-4244-4124-2/10/$25.00 ©2010 IEEE 674
The electrodes were integrated in an electrochemical cell,
constructed in PMMA using a numeric control device from a
CAD layout. Syringes were employed for the supply of buffer
B. Electronic instrumentation
A portable potentiostat was developed to allow
point-of-care measurements. The design can control the
reference electrode in the -2.5 V to 2.5 V range, allowing its
use for the present foot-and-mouth biosensor, as well as other
electrochemical biosensors. The allowed working electrode
current is in the -10 µA to 10 µA range. The circuit is shown in
The circuit uses a low offset operational amplifier (OpAmp)
to control the counter electrode (OP07). To avoid loading the
reference electrode an LT1056 OpAmp was selected,
providing an input impedance of 1012 ohms. The current to
voltage conversion, carried out in the working electrode
circuitry, was also implemented using an LT1056 OpAmp.
This OpAmp introduces a very low current error, typically in
the range of 10 pA, and guarantied to be under 0.34 nA for the
full operation range.
The potentiostat is controlled by a microcontroller
connected to a PC using the Universal Serial Bus (USB). The
microcontroller has a 10 bits A/D converter, used to measure
the working electrode current, and a PWM output, used to
control the reference voltage.
C. Immobilization of enzymes and antigens onto the
3ABC, a non-structural protein from the foot-and-mouth
disease virus, was immobilized on screen printed Au
electrodes employing cysteine and a carbodiimide as
molecular linkers between the gold electrode and the protein.
Electrodes were cleaned with a H2SO4 : H2O2 30% (2:1)
solution and thoroughly rinsed with water (miliQ quality),
immersed overnight in a solution containing 40 mM
3-mercaptopropionic acid and 75 % ethanol + 25% water.
The electrodes were treated during 30 min with 20 µl of a
solution containing 0.1 M 1-ethyl-3(3-dimethyl aminopropyl)
carbodiimide and 25mM N-hydroxysuccinimide in 0.1 M
PBS buffer of pH 7, and then 20 µl of 3ABC 0.22 µg/µl, also
in a 0.1M PBS buffer of pH 7, were added onto each
electrode. After 45 min, the electrodes were rinsed again with
high quality water and immersed overnight in quenching
buffer (0.262% glycine an 1% gelatine). Then the electrodes
were rinsed again and ready to be used.
For the serologic tests, 3ABC coated electrodes were
incubated with different sera during one hour and rinsed with
0.1% Tween 20 in PBS buffer by means of a controlled flux
syringes. Then the electrodes were incubated with anti-Ig
conjugates during an hour, rinsed with 0.1% Tween 20 in PBS
buffer and finally the electrochemical measurements were
Fig. 3. Potentiostat circuit.
D. Electrochemical measurements
Electrode potential was changed from 0 to –300 mV
applying 50 mV steps 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 to
1.5 mM. AgAgCl was employed as a reference electrode (a
wire of 1 mm diameter).
In the case of electrochemical immunodetectors, an antigen
(3ABC proteins in this case) is bonded to the electrode
surface. If this electrode is placed in contact with a serum
containing 3ABC antibodies (i.e. a “positive serum”),
antigen-antibody complexes are formed, which can further be
recognized by a second antibody. The second antibody is
labeled with an HRP. Thus, an antigen-antibody-
(HRP-labeled antiantibody) complex is formed on the
electrode. The activity of the HRP can be electrochemically
detected after addition of hydrogen peroxidase and a suitable
redox mediator, in a similar fashion as in the case of enzymatic
electrodes . This process is schematically shown in Fig. 4.
Fig. 4. Schematic representation of enzyme-linked immuno assay with
On the other hand, if a 3ABC coated electrode is placed into
contact with a serum without the presence of 3ABC antibodies
(a “negative serum”), no antigen-antibody complexes are
formed and, consequently, no HRP activity is
electrochemically detected. It is important to bear in mind that
3ABC is non structural protein, while vaccines for the
foot-and-mouth disease are based on structural proteins,
without 3ABC. Therefore, infected cattle produce antibodies
against 3ABC, while vaccinated cattle do not produce
anti-3ABC antibodies. This fact allows a differentiation
between vaccinated cattle and infected cattle, as the former
give a positive result with this test while the second give a
Figure 5 shows the current-potential curves obtained for a
positive and a negative serum. The difference in current
values is high enough as to permit the discrimination of
infected and non-infected specimens from this
electrochemical enzyme-linked immunoassay.
Figure 6 shows the measurement results for different sera.
Abscissa axis represents each serum with its code
identification, while y-axis represents current (nA) results,
normalized to the serum number 325.
-300 -250 -200 -150 -100 -50 0
electrode potential (mV)
Fig. 5. Current.potential curves obtained under steady state conditions for an
enzyme-linked immuno assay for a negative serum (squares) and a positive
serum (circles). Hydrogen peroxide concentration was 1.5 mM.
325 752 4 915 0 1 2 6 1256 1257 1258 1259 1260
Fig. 6. Results for positive (infected) serum, negative (normal) and
vaccinated serum, normalized to #325 serum.
The development of an inmunosensor for the point-of-care
detection of the foot-and-mouth disease was presented. The
detection method, based on an electrochemical ELISA test,
proved to be as sensible as the standard fluorimetric method.
The serum of infected cattle was clearly differentiated from
those of vaccinated and non-infected cattle. The detection
system is small, portable and specially suited for the detection
of the disease in remote and harsh environments.
 A.J. Bard, L.R. Faulkner, “Electrochemical Methods: Fundamentals
and Applications”, John Wiley & Sons, 2001.
 S.Campuzano, M.Pedrero, J. Pingaron, “A peroxidase-TTF biosensor
based on self-assembled monolayer modified Au electrodes for the
flow-injection determination of hydrogen peroxide”, Talanta 66 (2005)
 María E. Ribone, María S. Belluzo, Daniela Pagani, Iván S. Macipar,
Claudia M. Lagier, “Amperometric bioelectrode for specific human
immunoglobulin G determination: Optimization of the method to
diagnose American tripanosomiasis”, Analytical Biochemistry 350
 G. Longinotti, P. Lloret, G. Ybarra, C. Moina, L. Hermida, O. Milano,
M. Roberti, L. Malatto, L. Fraigi, L. “Screen-printed electrochemical
biosensors based on magnetic core-shell nanoparticles,” Proceedings of
6th Ibero-American Congress on Sensors, IBERSENSOR 2008.