On-Site Detection of Multiple Hepatotoxic Pyrrolizidine Alkaloids in Real Samples Using Label-Free Fluorescent Aptazymes

Hepatotoxic pyrrolizidine alkaloids (HPAs) are environmental and foodborne toxins found in more than 6000 flower plants and are a serious health hazard to humans. Therefore, rapid and accurate on-site detection of HPAs during production, processing, and transportation is a preliminary method to reduce the exposition of HPAs. In this work, a label-free fluorescent aptazyme, named LFAzyme, has been developed for fast, cost-effective, and visual detection of HPAs in real samples. LFAzyme self-assembled by a recognition segment (RS) and a "helper" DNA (HD) has the ability to quench the fluorescence of rhodamine B (RhB) in the presence of hemin and H₂O₂. With the addition of HPAs, the target would preferentially bind to RS that contains the sequence of the aptamer, yielding the destruction of LFAzyme and the recovery of RhB fluorescence. This assay enabled naked-eye determination of HPAs as low as 0.1 μM within 5 min, and the whole analysis could be completed in 15 min. The limit of detection value of 43 nM was obtained using a microplate reader. Our sensor validation with spiked real samples, including tea and honey, exhibited satisfactory HPA recovery within the range of 97.00-103.15%. Our LFAzyme could potentially be used for the on-site and naked-eye detection of HPAs or other targets with available aptamers.