Rapid single-molecule detection of Salmonella DNA based on an electrode surface-based polymerase chain reaction technique: Original scientific paper

Micheal Okeke; Dipak Koirala; Mathar Bashir; Glen Soderquist; I. Francis Cheng

doi:10.5599/jese.3403

Authors

Micheal Okeke Department of Chemistry, University of Idaho, 875 Perimeter Dr, Moscow, ID-83844, USA https://orcid.org/0009-0006-7064-2780
Dipak Koirala Department of Chemistry, University of Idaho, 875 Perimeter Dr, Moscow, ID-83844, USA https://orcid.org/0000-0003-1731-2644
Mathar Bashir Department of Chemistry, University of Idaho, 875 Perimeter Dr, Moscow, ID-83844, USA https://orcid.org/0000-0003-4318-9846
Glen Soderquist Department of Chemistry, University of Idaho, 875 Perimeter Dr, Moscow, ID-83844, USA https://orcid.org/0009-0000-3053-7552
I. Francis Cheng Department of Chemistry, University of Idaho, 875 Perimeter Dr, MS 2343, Moscow, ID, 83844, USA https://orcid.org/0000-0003-3863-8168

DOI:

https://doi.org/10.5599/jese.3403

Keywords:

DNA amplicons detection, electrochemical biosensor, Salmonella detection, polymerase chain reaction electrode, single DNA molecule detection

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.

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Rapid single-molecule detection of Salmonella DNA based on an electrode surface-based polymerase chain reaction technique

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