A self-assembly monolayer (SAM) of hairpin DNA can be formed on a gold substrate in order to make special biosensors. Labeling the hairpin loop probes with electroactive compositions rather than a fluorophore illustrates interesting profiles of redox current versus temperature. For a biosensor interacting with perfectly complementary targets, the profile shows a characteristic plateau, which disappears when the targets have a single base variation. The plateau is split into multiple steps by tuning the hybridization temperature. We propose that the phenomena are due to hairpin loop compartmentalization. The novel characteristics lead to a thermal gradient detection method that permits perfect discrimination of a target sequence from single nucleotide mismatches.