Temperature-Enhanced Colistin Resistance Gene Detection with Electrochemical Impedance Spectroscopy Biosensors.

Antibiotic resistance is now one of the biggest threats humankind is facing, as highlighted in a declaration by the General Assembly of the United Nations in 2016. In particular, the growing resistance rates of Gram-negative bacteria cause increasing concerns. The occurrence of the easily transferable, plasmid-encoded colistin resistance gene further worsened the situation, significantly enhancing the risk of the occurrence of pan-resistant bacteria. There is therefore a strong demand for new rapid molecular diagnostic tests for the detection of gene-associated colistin resistance. Electrochemical impedance spectroscopy (EIS) is a well-suited method for rapid antimicrobial resistance detection as it enables rapid, label-free target detection in a cost-efficient manner. Here, we describe the development of an EIS-based gene detection test, including the design of -specific peptide nucleic acid probes and assay specificity optimization through temperature-controlled real-time kinetic EIS measurements. A new flow cell measurement setup enabled for the first time detailed real-time, kinetic temperature-controlled hybridization and dehybridization studies of EIS-based nucleic acid biosensors. The temperature-controlled EIS setup allowed single-nucleotide polymorphism discrimination. Target hybridization at 60 °C enhanced the perfect match/mismatch (PM/MM) discrimination ratio from 2.1 at room temperature to 3.4. A hybridization and washing temperature of 55 °C further increased the PM/MM discrimination ratio to 5.7 by diminishing the mismatch signal during the washing step while keeping the perfect match signal. This newly developed gene detection test enabled the direct, specific label, and amplification-free detection of gene harboring plasmids from .