Signal-off tuned signal-on (SF-T-SN) colorimetric immunoassay for amantadine using activity-metalmodulated peroxidase-mimicking nanozyme

Immunoassays facilitate a wide range of rapid, simple and sensitive biochemical detection analyses. We developed a signal-off tuned signal-on (SF-T-SN) colorimetric immunoassay for the detection of the antiviral drug, amantadine (AMD). Using this strategy, the conventional competitive signal-off immunoassay acts as a foundation to induce different immune recognition events in the absence and presence of AMD. The introduction of activity-metal modulated peroxidase-mimicking nanozymes converts the conventional signal-off model to a signal-on model, thereby facilitating drug detection, and overcoming disadvantages with natural enzymes. Importantly, due to switching of the reporting mechanism, and the high catalytic activity of the nanozyme, this “off-to-on” pathway makes the nanozyme-based colorimetric signal proportional to AMD concentrations, thereby circumventing the inherent drawbacks of limited signaling abilities and sensitivities with conventional signal-off immunoassay-based small molecule detection systems. AMD concentrations can be read by the eye or by the optical signals. Based on this protocol, system sensitivity (0.195 ng mL −1 AMD for the naked eye, and 0.134 ng mL −1 AMD for optical detection) not only outperformed reported methods, but it dramatically improved the naked eye observations and UV–vis measurements, when compared with conventional signal-off immunoassays. AMD concentrations spiked into chicken samples were also detected with acceptable recoveries, and relative standard derivations (RSD), indicating this method is highly applicable for biochemical analyses and food safety monitoring.