Label-free and enzyme-free fluorescent biosensor for ultrasensitive detection of tobramycin via cascade signal amplification.
Uncontrolled use of tobramycin (TOB) raises serious health concerns, including nephrotoxicity and ototoxicity, making it imperative to develop simple and sensitive detection methods. This study introduces a novel enzyme-free and label-free fluorescent biosensor based on a cascade signal amplification strategy, integrating an entropy-driven DNA circuit (EDC) with catalytic hairpin assembly (CHA). The mechanism is initiated by the specific binding of TOB to its aptamer, which induces a conformational change and exposes a priming sequence. This sequence triggers a strand displacement reaction to release an initiator strand, subsequently activating the CHA process to generate fuel strands. These fuel strands drive the EDC, resulting in the liberation of numerous G-quadruplex sequences from the stem of hairpin probes. The released G-quadruplexes bind to thioflavin T (ThT), yielding a significantly enhanced fluorescence signal proportional to the TOB concentration. The proposed biosensor exhibits outstanding analytical performance with a detection limit of 92 fM and a wide linear range from 3 pM to 30 nM. Furthermore, the method effectively distinguishes TOB from other interfering antibiotics, highlighting its high specificity. It also demonstrates practical applicability by achieving satisfactory recovery rates in milk sample assays. With its high sensitivity, cost-effectiveness, and operational simplicity without the need for complex enzymes or labels, the proposed strategy offers a promising platform for food safety supervision and clinical diagnostics.