Pathogen-Specific Electrochemical Real-Time LAMP Detection Using Universal Solid-Phase Probes on Carbon Electrodes

Abstract

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

Topics & Concepts