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.
A novel triple-modal biosensor integrating hybridization chain reaction (HCR) with CRISPR/Cas12a was developed for sensitive and selective detection of kanamycin (KANA) and bisphenol A (BPA). The system employs a phosphorothioate-modified G-rich hairpin (SHG4-2) as a dual-functional reporter probe, which resists Cas12a trans-cleavage and enables multimodal signal output via SG-quadruplex (SG4) formation. Upon target recognition by aptamers, an initiator strand is released to trigger HCR amplification, generating long double-stranded DNA products that activate Cas12a trans-cleavage. This cleaves the (SHG4-2) probe, releasing SG-rich sequences that self-assemble into SG4 structures, yielding fluorescence (with Thioflavin T), colorimetric (via SG4/hemin-catalyzed TMB oxidation), and smartphone-readable RGB signals. This platform enables parallel detection of a single target analyte, allowing flexible detection modes. Under optimized conditions, the sensor achieved detection limits as low as 20.1 pM for KANA and 6.8 pM for BPA in fluorescence mode, 32.3 pM for KANA and 17.1 pM for BPA in colorimetric mode, and 74.3 pM for KANA and 35.8 pM for BPA in smartphone mode, with excellent selectivity against interfering analogues. Successful application in spiked milk samples demonstrated high recovery rates and good reproducibility. In addition, the platform supports the logical gate operations of OR (single-target detection) and AND (dual-target detection), which allows flexible detection modes. This work presents a versatile, amplification-enhanced multimodal sensing strategy for environmental and food safety monitoring, highlighting its potential for logic-driven biosensing applications.
Xiaolong Li, Daxiu Li, Yanni Wang et al.· Analytical Methods· 0 citations
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