A new selective electrochemical genosensor has been developed for the detection of an 86-mer DNA peanut sequence encoding part of the allergen Ara h 2 (conglutin-homolog protein). The method is based on a sandwich format, which presents two advantages: it permits shortening the capture probe and avoids labeling of the target. Screen-printed gold electrodes have been used as platform for the immobilization of oligonucleotides by the well-known S-Au bond. Mixed self-assembled monolayers (SAM), including thiol-modified capture probe and mercaptohexanol, were prepared to achieve an organized, homogeneous and not too compact SAM in which unspecific adsorption of the capture probe would be prevented. The optimization of the sensing phase was carried out using the Design of Experiments (DoE) approach. Traditionally, response optimization is achieved by changing the value of one factor at a time until there is no further improvement. However, DoE involves regulating the important factors so that the result becomes optimal. Optimized conditions were found to be 1.34 µM for capture probe concentration and 3.15 mM for mercaptohexanol (spacer) concentration. When the optimal conditions were employed the analytical performance of the proposed genosensor improved significantly, showing a sensitivity as high as 3 µA/nM, with a linear range from 5×10(-11) to 5×10(-8) M and a detection limit of 10 pM.
Keywords: Ara h 2 peanut allergen; Design of Experiments; Electrochemical genosensor; Self-assembled monolayers.
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