Sequence-specific electrochemical detection of enzymatic amplification products of Salmonella genome on ITO electrodes improves pathogen detection to the single copy level

Abstract

New insights into rational designing electrochemical genosensors for sequencespecific detection of enzymatic nucleic acid amplification products. Rules for the adaptation of PCR primers to isothermal helicase dependent amplification facilitating its adoption for point-of-need applications.  Electrochemical genosensor paired to PCR achieves single copy detection of Salmonella genome  Under isothermal conditions, helicase dependent amplification-electrochemical genosensor improves in 2-fold the detectability of real-time PCR *Research Highlights Sequence-specific electrochemical detection of enzymatic amplification products of Salmonella genome on ITO electrodes improves pathogen detection to the single copy level

Biosensors Based on Isothermal DNA Amplification for Bacterial Detection in Food Safety and Environmental Monitoring

“…HDA was used for the amplification of typA [ 36 ] and bipA [ 37 ] genes of Salmonella spp. These genes are involved in the virulence and infectivity of the pathogen and are less subjected to spontaneous silent mutations than the gene invA , which most of the Salmonella spp.…”

“…DNA amplification assays address. Although PCR primers may be used in HDA, they normally require more restrictive conditions (e.g., length between 24–33 bp), so they are normally modified to obtain the HDA ones [ 37 ]. In one of their works, Barreda-García and collaborators [ 36 ] immobilised thiolated primers to indium tin oxide (ITO) electrodes and solid-phase amplification took place.…”

“…DPV was used to measure the 1-naphthol produced by the reaction of the enzyme alkaline phosphatase (ALP) with the substrate 1-naphthyl phosphate ( Figure 4 ). In another work [ 37 ], the authors used a similar strategy but performed an exhaustive optimisation of several parameters, including the amount of capture probe immobilised on the electrode surface, the conditions of the immobilisation reaction, and the incorporation of a thermal treatment step before amplification to avoid secondary structures, among others. The HDA reaction was carried out in solution followed by a sandwich hybridisation assay using a capture probe immobilised on an ITO electrode and a FITC-labelled reporter probe for the subsequent interaction with the anti-FITC antibody-ALP conjugate.…”

DNA/RNA Electrochemical Biosensing Devices a Future Replacement of PCR Methods for a Fast Epidemic Containment

“…Helicase dependent amplification (HDA) is another widely employed isothermal system for amplification of target DNA [ 81 ]. Helicase is being used to unwind the double-stranded DNA instead of temperature-dependent denaturation during polymerase mediated amplification of the target gene [ 82 ]. Barreda-García et al reported an asymmetric HDA for that resulted in a single-stranded target DNA from Mycobacterium tuberculosis [ 69 ].…”

A review of microfabricated electrochemical biosensors for DNA detection

“…They then demonstrate a combination of electrochemical and optical measurement techniques by characterizing DNA hybridization on a silane anchored SAM, using Fourier Transform Infra-Red Spectroscopy at the same time as cyclic voltammetry. The advantages of ITO are also utilized by Barreda-Garcia et al, who developed an optimized biosensor for detecting Salmonella (Barreda- García et al, 2017García et al, , 2018. They demonstrate isothermal gene amplification on the same surface as their sensor and were able to detect single molecules of the target.…”