This study reports on a novel biosensor for K-ras mutation gene detection by using hairpin locked nucleic acids (LNA) probes dually labeled with amines groups and biotin. The hairpin LNA probes was covalently immobilized on poly-Eriochrome cyanine R (ECR) film modified glassy carbon electrode (GCE) as a switch. Streptavidin-horseradish Peroxidase labeled gold nanoparticles (SA-HRP-AuNPs) bioconjugate were used as tracer for signal amplification. The immobilized hairpin LNA probe was in the "closed" state in the absence of K-ras mutation gene, which shielded biotin from being approached by the bulky SA-HRP-AuNPs bioconjugate due to the steric effect. When hairpin LNA probes hybridized with K-ras mutation gene, hairpin LNA probes undergo significant conformational change, forcing biotin away from GCE surface. As a result, the biotin label becomes accessible by the SA-HRP-AuNPs bioconjugate, and K-ras mutation gene hybridization event can be sensitively transduced via the numerous HRP enzymatically amplified electrochemical current signal. This new biosensor had a good specificity to distinguish the K-ras mutations, K-ras wild-type sequence, and a high sensitivity. (C) 2013 Elsevier Ltd. All rights reserved.