Development of one-step magnetic assay for the detection of fentanyl

Abstract

This study introduces a novel one-step magnetic immunoassay (mMINA) designed for effective and sensitive detection of fentanyl, a potent synthetic opioid. The proposed method offers several advantages over the standard ELISA protocol, notably a substantial reduction in procedural steps from sample application to analysis. Simplicity directly translates into saved time, lowered measurement costs, minimized potential for errors, and enhanced user-friendliness and precision. The assay utilizes 96-well magnetic microplates for solid-phase separation, fluorescently labeled anti-fentanyl antibodies, and fentanyl-functionalized iron oxide nanoparticles as competing probes. Upon co-incubation, free fentanyl in the sample competes with nanoparticle-bound fentanyl for limited antibody binding, resulting in an inversely proportional fluorescence signal. The method demonstrated high specificity and sensitivity, with a limit of detection (LoD) of 1.562 nM in blood plasma, and showed high selectivity with structurally similar opioids such as morphine, heroin, or cocaine. It maintained performance in complex biological matrices, underscoring its robustness and reproducibility. Importantly, the modular platform design, based on commercially available antibodies, allows adaptation to other small-molecule targets. This flexibility, combined with operational simplicity and low cost, positions the mMINA assay as a practical tool for rapid drug screening in clinical, forensic, and field-based settings.