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Development and Characterization of a Piezoelectric Immunosensor for Determination of Domoic
Acid in Food Samples
Summary
In the present study a label-free piezoelectric crystal immunosensor,
based on an indirect competitive assay format, for the detection of
domoic acid (DA) in shellfish is described. After physical absorption
of the conjugate on the gold surface of the quartz crystals a competition
step was performed by putting in contact with the crystal a solution
containing the specific polyclonal antibody against domoic acid
and the DA as free toxin. The build up of mass on the crystal surface,
registered as a frequency decrease by the quartz crystal microbalance,
can be related to the amount of free toxin in the solution in
the 0–10 µg/g range. The sensor is capable to measure domoic acid
at the established regulatory level (20 µg/g) after a 1:4 (v/v) dilution
of the sample extract. The repeatability obtained by using three different
crystals was lower than 7%. Regeneration assays have shown
the possibility to reuse the modified surface by using 0.1 M NaOH as
dissociation agent. The sensor could have application as screening
method for compliance with European food legislation.
Zusammenfassung
Diese Arbeit beschreibt eine Methode zur Bestimmung von Domoinsäure
(DA) in Schalentieren. Eingesetzt wurde ein piezoelekrischer
Immunsensor, der auf einem indirekten kompetitiven Assay basierte.
Zuerst wurde die Goldoberfläche des Kristalls mit einem Protein-DA-
Konjugat beladen und anschließend in eine Lösung aus polyklonalem
DA-Antikörper und freier Domoinsäure gegeben. Die Massenveränderung
an der Kristalloberfläche führte zu einer Resonanzfrequenz-
Verschiebung des Quarzkristalls. Aus dieser Verschiebung konnte
die Menge an freiem Toxin in der Lösung bestimmt werden (Bereich:
0–10 µg/g). Mit diesem Sensor war es möglich die DA-Konzentration
im Bereich der zulässigen Höchstmenge von 20 µg/g nach einer
1:4-Verdünnung der Probeextrakte zu bestimmen.
Introduction
Domoic acid (DA) is a neuroexcitatory toxin that has been
identified in marine samples. This neurotoxic amino acid is
responsible of amnesic shellfish poisoning, which includes
gastrointestinal and neurological disorders. For human consumption,
a maximum content of 20 mg/kg of DA was established
in the Commission Decision 2002/226/EC 1) . Because
of the occurrence of this natural toxin is so widespread,
there is an urgent need to develop simple and sensitive methods
that can be used for quality control in order to guarantee
the food safety for the consumer.
The techniques most extensively used for that purpose are
based on liquid chromatography with UV and fluorescence
DLR | November/Dezember 2008 «
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Ana Rodríguez #1,2 , Josefa Barrero1 , Andri Papadopoulou1 and
Francois Rossi1 1 European Commission, Joint Research Centre, Institute for Health
and Consumer Protection, Via E. Fermi 1, I-21020 ISPRA (VA) Italy
2 University of Santiago de Compostela, Santiago de Compostela E-
15782, Spain
detection; although these methods present high sensitivity
they are time-consuming and require expensive instrumentation
2,3) . In recent years, the rapid detection methods
are gaining popularity and acceptance as a simple alternative,
since they are a very useful as screening tools and
excellent complement to confirmatory methods. Several
enzyme-linked immunosorbent assays (ELISA) for domoic
acid have been reported in the literature 2,4–6) however
other methods such as electrochemical immunosensors,
for determining the toxin, based on screen-printed electrodes
have also been described 3,7) . Another technique
suitable for immunoanalysis of DA is based on the Quartz
Crystal Microbalance (QCM), a sensitive acoustic technique
that allows the measurement of resonance frequency
shifts attributed to changes in the total oscillating
mass of a piezoelectric crystal. In these systems a shift in
the frequency of the quartz crystal, registered during the
antigen-antibody reaction, is converted to a mass change
on the crystal surface determined using the Sauerbrey equation
8) (*).
(*) ∆m = –
c ∆f
n
where, C = 17.7 ng Hz -1 cm -2 for a 5 MHz quartz crystal,
n = 1, 3, 5, 7 is the overtone number.
Piezoelectric transducers, being high sensitive direct mass
sensors 9) , allow applying label-free techniques in immunoassays
which are known to be advantageous in terms of
simplicity of performance and rapidity. These systems have
been successfully applied in different areas; analysis of contaminants
10,11) , evaluation of protein immobilization 12) , nucleic
acid sensors 13) , clinical analysis 14) .
In this study, the development and characterization of a
piezoelectric sensor for determination of domoic acid on
the basis of a competitive assay is reported.
# Dr. Ana Rodriquez, phone: +34981598450, Fax: +34981594912,
e-mail: anarbq@usc.es
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