Pathogen-Specific\nElectrochemical Real-Time LAMP Detection\nUsing Universal Solid-Phase Probes on Carbon Electrodes

Epidemic infections and spreading antibiotic resistance\nrequire\ndiagnostic tests that can be rapidly adopted. To reduce the usually\ntime-consuming adaptation of molecular diagnostic tests to changing\ntargets, we propose the novel approach of a repurposable sensing electrode\nfunctionalization with a universal, target-independent oligonucleotide\nprobe. In the liquid phase covering the electrode, the target sequence\nis amplified by MD LAMP (mediator-displacement loop-mediated isothermal\namplification) releasing a generic methylene blue-labeled mediator,\nwhich specifically hybridizes to the solid-phase probe. To demonstrate\nthe universality of the approach, two different pathogens, Staphylococcus aureus (crude lysate) and Treponema pallidum, are detected using the same solid-phase\nprobe. The reactions reach a limit of detection of 1 × 10<sup>3</sup> and 4 × 10<sup>2</sup> copies per reaction within 30\nmin, respectively. The solid-phase probes carry a carboxymethyl aniline\nmodification to form covalent C–C bonds on low-cost carbon\nelectrodes. Maximum surface coverage and maximum hybridization signals\nare observed at grafting concentrations of ≥2 μM solid-phase\nprobes. Successful detection of spiked target DNA in real swab samples\nand with three different commercial amplification buffers proved the\nbroad applicability of this assay approach. The electrochemical MD\nLAMP is fast, compatible with dsDNA targets, and requires only minimal\nadaptation of an established amplification method. It is easily transferable\nto existing analytical electrochemical platforms, allowing the consumable\nto be synergistically used for different targets. The suggested approach\nof repurposable functionalized electrodes can also be considered to\nincrease the preparedness for future epidemic or pandemic outbreaks\nas well as rapidly evolving resistance patterns or variants.