<strong>Electrochemical Detection of <em>Salmonella</em> via On-surface Isothermal Amplification of its Genetic Material onto Highly Stable and Reproducible Indium Tin Oxide Platforms</strong>

Abstract

An on-surface isothermal helicase-dependent amplification is devised for simple, point-of-need quantification of bacterial genomes. The method relies on the enzyme-extension of a thiol-modified reverse primer anchored to indium tin oxide electrodes, which shows strikingly high thermal and storage stability. Amplification and electrochemical detection of only 10 genomes are thus performed on the same platform without thermal cycling.Sequence-specific nucleic acid-based sensors hold great economic potential for decentralized genetic testing, a key requirement not only for clinical diagnostics but also in biosafety or food safety control and environmental monitoring. Electrochemical devices are particularly suitable for satisfying most of the characteristics for an ideal point-of-need molecular test.1,2 Despite very low limits of detection being provided, 3-5 the electrochemical DNA sensors reported until now are most often tested using target short oligonucleotides, or sequences obtained after polymerase chain reaction (PCR) amplification. These hybridization-based platforms for DNA detection are challenging to deploy in genomic DNA where the efficiency of hybridization is hampered by its large size and complexity. Consequently, the development of an integrated and miniaturized platform for genomic DNA quantification usually requires a combination of a sample pretreatment step, 6,7 uncoiling and cutting the genome, and a sequence-specific detection method. One approach is to integrate a nucleic acid-based sensor and an amplification method, which contributes not only to restricting the size but also to multiplying the target genome. PCR is the gold standard amplification technique, although the need for thermal cycling limits its use in easy-to-use devices for on-site detection. 8Alternative amplification methods that do not require heating and cooling have been developed over the last two decades. In these technologies the amplification reaction takes place at a constant temperature, usually higher than 37 1C, by adapting enzymatic mechanisms from natural biological processes. 8,9 However, to truly integrate isothermal amplification and electrochemical detection there still exist important hurdles to overcome, mainly reproducibility and thermal stability of the sensing phase. Gold surfaces functionalized with DNA oligonucleotides through the formation of thiol-based self-assembled monolayers (SAMs) are a common approach for electrochemical genosensing, even though the poor stability under dry storage conditions and thermal stability in aqueous solutions limit their widespread commercial application. 10 The use of alternative surfaces for DNA immobilization remains little explored. Indium tin oxide (ITO), a transparent semiconductor material that can be used both as a support for oligonucleotide immobilization and as a resistive heater, has been only scarcely used for DNA immobilization. 11,12Herein, we demonstrate that a simple and general approach to covalently immobilize thiol-modified oligonucleotides on ITO s...