Fabrication of a Near-Infrared Upconversion Nanosensor for the Ultrasensitive Detection of eARGs Using a Dual-Amplification Strategy

Extracellular antibiotic resistance genes (eARGs) have emerged as significant environmental contaminants due to their role in the transmission and proliferation of antibiotic-resistant bacteria, posing a major threat to global health. Traditional detection methods for eARGs require complicated equipment, lengthy analysis times, and relatively low selectivity. Furthermore, eARGs are present in low concentrations in surface water samples, which presents considerable challenges to the sensitivity of detection assays. Therefore, there is an urgent need to develop more accessible, stable, and sensitive detection methods. In this work, we developed an ultrasensitive upconversion nanosensor utilizing a dual-amplification strategy for the detection of trace eARGs (bla-TEM). The upconversion nanosensor was activated upon the capture of bla-TEM and subsequently enriched through magnetic separation. Following this, a cascade nicking-polymerization amplification process occurred in a single reaction facilitated by a magnetic capture probe, an upconversion recognition probe, and the relevant enzymes. The upconversion nanosensor functions as both the direct target-recognizing moieties and signal reporters, replacing the energy donor in conventional luminescence resonance energy transfer-based upconversion nanosensors. Ultimately, the strategy demonstrated excellent sensitivity with a limit of detection (LOD) of 0.093 aM, rapid detection in less than one hour, good selectivity, and high accuracy compared to conventional polymerase chain reaction (PCR) assays. These findings provide valuable insights for the development of ultrasensitive detection assays for emerging environmental pollutants.