Abstract
There is a current need for rapid, selective and sensitive specific detection of Salmonella in food matrices. We demonstrate a technique for rapid single-molecule detection of Salmonella DNA based on an electrode surface-based polymerase chain reaction (PCR) based sensing of 48-base target single-stranded DNA (ssDNA) (CP014358.1 GenBank), chicken-juice matrix under 20 minutes. This is based on the covalent attachment of a probe ssDNA to pseudo-graphite. This sensor can be used in two different modes, without and with PCR, for lower limits of detection (LOD). The pseudo-graphite electrode is prepared for ssDNA attachment through the functionalization of its surface with carboxylate groups, which form amide linkages with amine-modified ssDNA. Without PCR, the probe-modified electrode is immersed in a sample complementary target matrix for 10 minutes, removed, and then placed into a solution of the double-stranded DNA (dsDNA) intercalator, cobalt(III) tris-phenanthroline. Detection of dsDNA formation between the probe and target sequences is achieved via the SWV of the surface-bound cobalt complex. The LOD of this method is 2.0 aM for the target within 12 minutes. Lower LOD’s are obtained by conducting PCR on the electrode surface. The on-electrode (oE) PCR process uses a 20-base primer attached to the pseudo-graphite. A chicken-juice sample matrix containing target ssDNA (48 bases of Salmonella typhimurium) was directly added to a PCR master mix with a reverse primer, placed in a standard PCR vial, and thermocycled 5 times. The electrode with surface-bound dsDNA was subjected to the cobalt(III) tris-phenanthroline protocol with a LOD of 35 zM. This is one DNA molecule within 17 minutes in a complex chicken-juice matrix.