A strategy to design DNA-biosensors by grafting probe-target duplex onto a 27 MHz quartz crystal microbalance gold surface is presented in this work. The idea that removing the DNA target after duplex grafting yields to a well accessible probe, and therefore to efficient target recognition, is investigated. Unfortunately, hybridization efficiency measured using such a straightaway protocol is equal to 15%, which is feeble by comparison with the 31% hybridization efficiency measured for biosensors designed with single-stranded probe. Successive DNA biosensors are designed in this work, taking into account phenomena that occur specifically in an interfacial environment, like non specific adsorption or exchange reactions between adsorbed molecules. A DNA-biosensor with an optimized biolayer in terms of probe accessibility and probe surface density is in fine designed: a 59% hybridization efficiency, for a complementary target including a 20-base non complementary sequence oriented toward the surface, is obtained for the biosensor designed with target-probe duplex. This hybridization efficiency is 3 times to 18% measured for biosensors designed with single-stranded probe. (C) 2012 Elsevier B.V. All rights reserved.