Beyond membrane integrity: Dielectrophoretic discrimination of physiological states in heat-stressed Campylobacter...

The viable but non-culturable (VBNC) state of Campylobacter jejuni complicates food safety monitoring, as current assays such as propidium monoazide quantitative PCR (PMA-qPCR) rely on membrane integrity and can overestimate the viability of physiologically compromised cells. This study evaluates a dielectrophoresis (DEP)-based microfluidic method as a rapid, label-free approach for characterizing heat-stressed C. jejuni. We established selective DEP capture conditions (≥4.0 MHz) that distinguished VBNC-candidate cells (defined here as culture-negative but membrane-intact) from autoclaved dead controls. DEP-based enumeration agreed with PMA-qPCR at moderate temperatures (50-60 °C), whereas a critical decoupling emerged at elevated temperatures (≥70 °C): DEP responsiveness markedly diminished despite persistent PMA-qPCR signals, indicating a loss of electrical polarizability in cells that retained membrane impermeability. In parallel, the number of proteins identified by LC-MS/MS declined with treatment severity, with a pronounced loss of respiratory-chain and ATP-synthesis components; this pattern is consistent with, but does not directly demonstrate, a temperature-dependent decline in energy-generating capacity. Together, these results show that DEP responsiveness and membrane permeability report distinct and only partially overlapping physiological states, and that DEP enables label-free capture and recovery of the electrically responsive subpopulation. Importantly, DEP responsiveness should be interpreted as an electrical-functional phenotype rather than direct proof of cellular viability; thus, rather than serving as a stand-alone viability assay, DEP provides information complementary to membrane-permeability-based methods for characterizing heat-stressed C. jejuni.