|Título/s:||Method and instrumentation of serologic diagnostics: An electrochemical enzyme-linked immunoassay for the diagnosis of mouth and foot disease|
|Autor/es:||Ciochinni, Andrés; Rey Serantes, Diego; Malatto, Laura; Roberti, Mariano; Tropea, Salvador; Fraigi, Liliana; Lloret, Paulina; Longinotti, Gloria; Ybarra, Gabriel; Moina, Carlos|
|Institución:||Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. UNSAM. Buenos Aires, AR |
INTI-Electrónica e Informática. Buenos Aires, AR
INTI-Procesos Superficiales. Buenos Aires, AR
|Palabras clave:||Inmunología; Inmunoensayos; Diagnóstico; Serología; Métodos electroquímicos; Detección; Detectores; Enfermedades; Enzimas; Anticuerpos; biosensores; Instrumentos electrónicos; Antígenos; Electrodos|
| Ver+/- |
Abstract—The development of an inmunosensor for the
point-of-care detection of the mouth and foot disease is
presented. The detector is based on an ELISA method with
amperometric detection. A non-structural protein, the 3ABC, is
used to selectively detect anti-3ABC antibodies. The biological
test is performed directly onto screen printed electrodes. A
dedicated small, portable potentiostat is employed for the control
of the sensors, as well as data acquisition 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 some sort 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 methods and instrumentation for an electrochemical
enzyme-linked immunoassay for the diagnosis of the mouth
and foot 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 proteinaceous 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 foot and mouth disease
II. EXPERIMENTAL DETAILS
A. Electrodes design and manufacture
A three-electrode cell was designed and constructed.
Ring-disk geometry was defined for the working and counter
electrodes. Disks with a diameter of 1000 µm were employed
*This work was supported by the Instituto Nacional de Tecnología
Industrial and the Instituto de Investigaciones Biotecnológicas.
as working electrodes, while counter electrodes were designed
to have ten times as much 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. Track
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) Matrix of 18 sensors. b) Photograph of a thick film
two-electrode configuration showing active circular area with 1000 µm
diameter for the working electrode.
The two thick film electrodes were printed onto α-Al2O3
substrates by conventional screen printing technology; the
third electrode was a silver chloride reference electrode.
Commercial Au organometallic paste (ESL D8083) 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. The electrodes were integrated in an
electrochemical cell, constructed in PMMA using a numeric
control device from a CAD layout.
B. Electronic instrumentation
A portable potentiostat was developed to allow
point-of-care measurements. The instrument can control the
reference electrode in the -2.5 V to +2.5 V range, allowing its
use for this and other biosensors. The allowed working
electrode current is in the -100 µA to +100 µA range.
The circuit uses a low offset operational amplifier (op-amp)
to control the counter electrode (OP07). To avoid loading the
reference electrode an LT1056 op-amp was selected,
providing an input impedance of 1012 ohms. The current to
Method and Instrumentation of Serologic Diagnostics: An
Electrochemical Enzyme-Linked Immunoassay for the Diagnosis of
Mouth and Foot Disease
Laura Malatto, Mariano Roberti,
Salvador Tropea and Liliana Fraigi
INTI – Electrónica e Informática
Paulina Lloret, Gloria Longinotti,
Gabriel Ybarra and Carlos Moina*
INTI - Procesos Superficiales
and Diego Rey Serantes
UNSAM - Instituto de
voltage conversion, carried out in the working electrode
circuitry, was also implemented using an LT1056 op-amp.
This op-amp 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 antigens onto the electrode surface
3ABC, a non-structural protein from the foot and mouth
disease virus, was immobilized on screen printed Au
electrodes employing 3-mercaptopropionic acid and a
carbodiimide as molecular linkers between the gold electrode
and the protein. Electrodes were cleaned with H2SO4:H2O2
30% (2:1) 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
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. A wire of 1 mm diameter of AgAgCl was employed
as a reference electrode.
In this electrochemical immunodetectors, an antigen
(3ABC) is linked to the electrode surface. If this electrode is
placed in contact with a serum containing anti-3ABC
antibodies (i.e. a “positive serum”), antigen-antibody
complexes are formed, which can be further recognized by a
second antibody. The second antibody is labeled with a redox
enzyme (horseradish peroxidase, i.e. HRP). Thus, an
antigen-antibody-(HRP-labeled anti-antibody) complex is
formed on the electrode. The activity of the HRP can be
electrochemically detected after the 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. 2.
On the other hand, if a 3ABC coated electrode is placed in
contact with a serum without 3ABC antibodies (a “negative
serum”), no antigen-antibody complexes are formed and,
consequently, no HRP activity is electrochemically detected.
Fig. 3 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.
Fig. 2. Schematic representation of enzyme-linked immunoassay with
-300 -250 -200 -150 -100 -50 0
electrode potential (mV)
Fig. 3. 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: 1.5 mM. The potential of
the working electrode was controlled with a potenciostat EG&G PAR 273A.
325 752 4 915 0 1 2 6 1256 1257 1258 1259 1260
Fig. 4. Results for positive (infected), negative (normal) and vaccinated sera,
normalized to serum number 325.
Fig. 4 shows the measurements results for different sera.
Abscissas represents each serum with its code identification,
while ordinates represent a magnitude proportional to the
measured current. This magnitude is corrected by the current
measured with an electrode without any immobilized
biomolecule and normalized to the current measured with the
serum number 325. High values for the ordinates account for
positive sera, while small values of the ordinates are obtained
for the negative and vaccinated sera.
The development of an immunosensor for point-of-care
detection of the mouth and foot disease was presented. The
detection method, based on an electrochemical ELISA test,
proved to be as sensible as the standard fluorimetric method.
The sera of infected cattle were clearly sorted out 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.
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