Authors: C. March, J.J. Manclús, A. Arnau, Y. Jiménez, T. Sogorb, A. Montoya

Event: 7th Workshop on Biosensors and Bioanalytical μ-Techniques in Environmental and Clinical Analysis. Kusadasi, Turkey, 10-14 September (2006)

 

Immunosensors are novel analytical devices in which the extreme selectivity provided by immunological interactions is combined with the high sensitivity achieved by electronic signal transducers. In the present work, the development of a Monoclonal Antibody-based Piezoelectric Immunosensor for the analysis of pesticides (carbaryl and the chlorpyrifos and triclopyr metabolite TCP) is described, and its main analytical characteristics are presented.

Specific carbaryl and TCP haptens were conjugated to a carrier protein (BSA) and then covalently attached to gold-coated AT-cut 9 MHz  quartz crystals, previously functionalized with a self-assembled alkanethiol (thioctic acid) monolayer. The activated piezoelectric crystal was inserted in a homemade Arnite cell that allowed only one face of the crystal to be in contact with the reagents. The cell was integrated in a flow-through system and pesticide detection was subsequently accomplished by competitive immunoassay in the conjugate-coated format. The assay consisted of a previous incubation of the analyte sample with its specific monoclonal antibody, followed by the immunoreaction of the analyte-antibody mixture with the hapten derivative immobilized on the sensor surface. The frequency variations of the piezoelectric crystal working in the microgravimetric mode were measured with a commercial Research Quartz Crystal Microbalance (RQCM) and correlated with the concentration of the respective pesticide in the sample. The total time required for a complete assay cycle, including regeneration, was 20 min.

As it was expected for binding inhibition immunoassays, the frequency signals provided by the sensor were inversely proportional to the pesticide concentrations. Sigmoidal calibration curves were obtained by fitting experimental points to a four-parameter logistic equation. The immunosensor sensitivity, expressed as the analyte concentration that reduced the assay signal by 50% (I₅₀) around 25 µg/l for carbaryl and 32 µg/l for TCP. The limits of detection, calculated as the pesticide concentrations that provide 90% of the maximum signal, were around 6 and 9 µg/l, respectively. This analytical performance would allow the detection of carbaryl and TCP in fruits and vegetables at European regulatory levels. For both pesticides, the hapten-functionalized piezoelectric crystals could be reused for more than one-hundred times with only slight reductions of the maximum signal and without significant losses of sensitivity.