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.
