Power-free and sensitive on-site detection of a foodborne bacterial pathogen via confinement effect activated nanomachines-mediated amplification (CEANMA)

Widespread deployment of foodborne pathogen testing in low- and middle-income countries (LMICs) remains challenging due to unreliable electricity and scarce laboratory infrastructure. To address this issue, we developed a power-free, sensitive, and visual biosensing assay, termed Confinement Effect Activated Nanomachines-Mediated Amplification (CEANMA), alongside a custom-designed portable device. CEANMA utilizes two DNA-engineered nanomachines capable of colocalizing on the target pathogen surface, thereby triggering proximity-induced rolling circle amplification (RCA). The resulting amplicons then activate the CRISPR/Cas12a to yield a bright fluorescence readout. The workflow requires only a single temperature control (42 °C). All reagents are formulated to allow 10-day room-temperature storage, eliminating dependence on cold-chain logistics. The palm-sized device (<200 g) integrates a chemically driven disposable heat pack and a hand-crank-powered LED module, entirely removing the need for external electricity. Device functionality remains stable across environmental temperatures of 15-35 °C and regardless of ambient-lighting variations. Representative foodborne bacterial pathogen, Salmonella enteritidis, was selected to valid the performance of the power-free CEANMA assay. As low as 16 CFU/mL of Salmonella enteritidis was accurately identified. Using our kit and portable device, minimally trained volunteers successfully detected target bacteria in food samples within 55 min. Given its simplicity, portability, and robust performance, the power-free CEANMA assay offers great potential for enhancing food safety surveillance and reducing the burden of foodborne illnesses in LMICs.