Resumen de Immunosensors based on interdigitated ¿-electrodes: application to pesticide detection
Taking into account the current need of low-cost methods, simplicity of use, fast and sensitive for the early determination of residues of pesticides in foodstuff, as well as the need of legislation that establish the limits of pesticides in wine, we decided to develop immunosensor devices which meet these needs. During this Thesis two novel immunosensors for quantification of small amount of free pesticide in red wine samples have been developed and demonstrated.
The immunosensors developed have been named: impedimetric and conductimetric immunosensor. Impedimetric immunosensor is based on an array of interdigitated ¿-electrodes (IDµE) and bioreagents specifically developed (antigen, antibody) to detect residues of pesticide, using unlabelled antibodies. Conductimetric immunosensor incorporates additionally non-specific antibodies labelled with gold nanoparticles. In both cases the reagents were covalently immobilized on the surface of the electrodes (interdigital space), and the biochemical determination of pesticide was possible without any redox mediator.
For the case of the impedimetric immunosensor, the detection method is based on impedimetric measurements (in a wide range of frequencies and at single frequency), whereas in the case of the conductimetric immunosensor the detection method is based on conductimetric measurements (DC measurements). The potential of the immunosensors to detected pesticides has been evaluated using atrazine in assay buffer as well as in red wine samples.
Impedimetric immunosensor does not require the use of any label to achieve the detection of small amounts (sub-ppb) of atrazine. On the other hand, conductimetric immunosensor offers the possibility of detecting small amounts (sub-ppb also) of atrazine by means of simple and inexpensive DC measurements. This opens the possibility of obtaining a commercial immunosensor of very easy use (do not require qualified personnel), transportable and economic.
Immunosensors fabrication includes the design, the transducer fabrication together to their functionalization. In this work, non-passivated interdigitated ¿-electrodes have been used as transducer. The sensors sensitivity is greatly increased thanks to the competitive immunoassay format where the impedimetric variation is related to the molecules of antibody, instead the molecules of pesticide (smaller than the antibody molecules).
An analysis of the impedimetric behaviour based on impedimetric spectroscopy techniques was conducted. From that was concluded that the devices developed are sensitive to changes in impedance caused by the bioreagents immobilized on the electrodes. The changes in the impedance were interpreted using an equivalent circuit which represents the system faithfully.
From the results shown in this work it becomes clear that residues of atrazine can be directly detected at sub-ppb concentrations, far below the Maximum Residue Level (MRL, 50 ¿g L-1) required by EC for residues of this herbicide in wine grapes. Thus is demonstrated that the matrix effect does not prevent the detection of atrazine using our devices.
As an additional approach, a flexible immunosensor was also developed. This flexible device is based on the use of a flexible interdigitated ¿-electrode (FIDµE), as well as in a flexible microfluidic platform. Currently the conductimetric immunosensors have also been demonstrated using FIDµE's. In this case, the LODs are around 2-3 ppbs. On the other hand, a prototype of a microfluidic platform was also designed and fabricated in order to transport the solutions, required for the assay, to the electrodes, avoiding the micropipettes. To date, the transport of liquids to the reservoir has been demonstrated. Then, we can conclude that this microfluidic system can be used as platform to the assay development, and therefore could be possible to obtain a lab-on-a-chip system.
