A reverse ELISA based on quantum dot-labeled TetR for rapid detection of tetracycline

The environmental and public health risks associated with the overuse of tetracycline (TC) antibiotics have garnered significant attention. Thus, rapid and accurate detection of these antibiotics is crucial both for evaluating their biochemical toxicity and for monitoring their misuse. In this study, a novel reverse ELISA assay that combines quantum dots (QDs) with an allosteric transcription factor (TetR) was developed for rapid TC detection, eliminating the need for incubation and signal amplification steps. Specifically, biotin-modified double-stranded DNA (dsDNA) was immobilized on a streptavidin-coated 96-well plate, after which QD-TetR conjugates were added. In the presence of TC, the QD-TetR conjugate binds TC and undergoes allosteric changes that result in its dissociation from the dsDNA. Method validation using seawater, sewage, milk, and honey samples showed a strong correlation ( R² = 0.9825) between fluorescence intensity and logarithmic TC concentrations (Y = −211.9X + 729.9). The assay demonstrated a detection limit of 0.02 μM with semi-quantitative visual detection (0.5-500 μM range) within 5 minutes, indicating strong potential for monitoring TC residues in environmental and food samples.