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.
High selectivity of genosensors is crucial for certain applications such as those involving species with high genetic variability. This is an unresolved problem when dealing with long target sequences that is further complicated when the target contains repetitive sequence domains. As a model for this situation, the problem of detecting gluten in food with identification of the source is studied. In order to discriminate the specific DNA sequence that encodes the wheat prolamin (gliadin) from rye and barley prolamins, the exquisite selectivity of a rationally designed hairpin capture probe is proposed and compared to a nonstructured capture probe. An electrochemical sandwich assay is proposed, involving capture probes chemisorbed on Au surfaces and biotinylated-signaling probes in combination with streptavidin-peroxidase labeling conjugates. As a result, a genosensor with similar sensitivity to that observed with linear probes but with complete specificity against closely related species was achieved. The surface-attached DNA stem-loop yields a device capable of accurately discriminating wheat DNA from rye and barley with a limit of detection of 1 nM.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.