Temperature-Enhanced mcr-1 Colistin Resistance Gene Detection with Electrochemical Impedance Spectroscopy Biosensors

Abstract

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 mcr-1 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 mcr-1 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 mcr-1 gene detection test, including the design of mcr-1-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 mcr-1 gene detection test enabled the direct, specific label, and amplification-free detection of mcr-1 gene harboring plasmids from Escherichia coli.

Temperature-Responsive Nanocarrier-Regulated Alternative Release of “Cargos” for a Multiplex Photoelectrochemical Bioassay of Antibiotic-Resistant Genes

“…Currently, novel PCR-free approaches for ARG detection have been developed, though scarce, such as optical sensor, − colorimetry, and electrochemical/photoelectrochemical (PEC) sensor. − Among these methods, the PEC assay has gained intensive attention, profiting from the different energy forms of the excitation and the detection signals with the merits of remarkable sensitivity, and inherent miniaturization. − The inherent maneuverability of the PEC assay, for example, the engineering of the photoactive materials, the flexibility of the applied excitation wavelength, and potential endows it with great promise as the multiplex assay. And thereafter the light/potentiometric-addressable or wavelength-resolved PEC multiplex assays have been established for multitarget analysis. − However, the processes for partitioning electrodes or fabricating electrode arrays on each tiny area are laborious and impair the sensitivity aroused from the insufficient utilization of light irradiation, limiting the development of multiplex PEC assays.…”

How to: screening for mcr-mediated resistance to colistin

“…From the other available molecular methods, the mcr-1 gene can be detected by an enzymefree homogenous electrochemical assay using the hybridization chain reaction and mcr-1specific toehold probe or by electrochemical impedance spectroscopy [59][60].…”