Gapped-duplex structure to label-free mismatch detection of pathogen DNA on solid substrate

• A gapped-duplex-based label-free DNA biosensor is developed.
• Horizontally aligned probe-DNA was capable of enhanced mismatch discrimination capability.
• This method has proved to have some advantages over conventional vertical hybridization protocol.

A label-free platform for DNA mismatch detection, that has the potential to overcome the major drawbacks of conventional microarrays, is proposed. We have designed a gapped-duplex-based approach for achieving horizontally aligned probe-DNA on a solid substrate that offers low steric hindrance with excellent mismatch discrimination capability, enhanced association constant, and a much faster response time (less than 15 min). The electron transfer characteristics of DNA sequences immobilized by proposed method on electrode surface are compared with those from conventional immobilization method. The potential of the method in discriminating single nucleotide polymorphism variants among Cryptosporidium parvum genotypes (e.g., human and bovine) is experimentally appraised.