The Development of a DNA Biosensor for the Detection of Klebsiella pneumoniae

This report addresses the development of a novel electrochemical biosensor for the detection of Klebsiella pneumoniae through the development of a surface modification strategy for boron-doped diamond and glassy carbon electrodes. Thiolated ssDNA probe strands, designed to capture the target of interest, were covalently attached to a diamond electrode surfaces through a 4 [(N-Boc)aminobenzene] diazonium linker, and to glassy carbon electrodes with N-Boc-1,2-diaminoethane before they were hybridized with a complementary target ssDNA strands. Using a redox active target strand, these attachments can be detected using cyclic voltammetry. The stability of the surface bound DNA was explored to demonstrate the commercial viability of the developed sensors. Modified BDD and glassy carbon electrodes were found to be more resistant to high temperatures than gold analogues when modified with DNA probes. The operational stability was examined using chronoamperometry and DNA bound to BDD and glassy carbon electrodes were found to resist reductive desorption, compared to typically used thiol-modified gold electrodes where the same DNA probe strand reductively desorbs. A novel DNA immobilization method which utilizes a combination linker/reported for BDD and graphene is also discussed. This new method exhibits excellent selectivity and durability, positioning itself as an innovative method for label free sensing. In addition, detection was achieved for artificial DNA as well as for the PCR products of live cell isolates using electrochemical impedance spectroscopy, and glassy carbon biosensors displayed increased sequence sensitivity compared to typical gold systems, with the ability to distinguish between single base-pair mismatches.