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An important factor affecting the performance of electrochemical genosensors is the immobilization strategy used to anchor the capture probes (CP) on the electrode surface. One of the most common immobilization systems involves the formation of self‐assembled monolayers of thiolated probes on gold surfaces, mainly due to its simplicity and effectiveness. Recent studies have shown that in two‐component thiolated DNA and mercaptohexanol (MCH) monolayers, there is incomplete backfilling that leads to defective surface blockage, producing unspecific background signal. In order to solve these problems, ternary monolayers have been proposed, in which besides MCH as primary diluent, a second diluent is added to enhance the hybridization efficiency and anti‐fouling properties of the surface. Herein, we report a thorough evaluation of different surface chemistries using two different sensors, one for the detection of a peanut allergen encoding sequence and the other one targeting a gluten‐encoding sequence. We assessed the relationship between CP length and the chemical characteristics of the diluents. Factors such as concentration, nature of the functional groups and chain length of the second diluent were evaluated. We found that length of diluents affects primarily the hybridization efficiency, while the type of functional groups, e.g. hydroxyl, carboxyl or a second thiol group, influences the degree of surface coverage. Results diverged between the two sensors evaluated.

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