Scanning electrochemical microscopy assay of DNA based on hairpin probe and enzymatic amplification biosensor

A novel scheme for scanning electrochemical microscopy (SECM) assay of DNA based on hairpin probe and enzymatic amplification biosensor was described. In this method, streptavidin-horseradish peroxidase (HRP) was captured by double-stranded DNA (ds-DNA) modified gold substrate via biotin-streptavidin interaction after hybridization of target DNA to the immobilized hairpin probe functioned with a biotin at its 3' end. In the presence of H2O2, hydroquinone (H2Q) was oxidized to benzoquinone (BQ) at the modified substrate surface through the HRP catalytic reaction, and the generated BQ corresponding to the amount of target DNA was reduced in solution by a SECM tip. The resulting reduction current allowed concentration detection of target DNA and SECM imaging of hybridization between the target DNA and the immobilized hairpin probe. The detection limit of this method was as low as 17 pM for complementary target DNA and it had good selectivity to discriminate between the complementary sequence and one containing base mismatches.