Aptamer-modulated haloperoxidase-mimicking CeO₂ nanozyme for multicolor and ratiometric colorimetric sensing

Colorimetric nanosensors based on nanozymes, particularly those utilizing a single absorption peak, often suffer from subtle color variations that challenge accurate visual interpretation and quantification. To overcome this constraint, a multicolor aptasensor by integrating CeO₂ nanozymes with haloperoxidase (HPO)-mimicking activity were developed for ratiometric colorimetric and visual sensing. The CeO₂ nanozymes with exceptional HPO-like activity catalyzes thymol blue iodination to induce dual absorbance peaks changes at 430 nm (decreasing) and 620 nm (increasing), resulting in clear yellow-to-blue color tonality discernible to the naked eye. Crucially, aptamer-regulated enzymatic inhibition is employed, where target binding triggers the release of complementary DNA to modulate the catalytic activity of CeO₂ nanozyme through charge-switchable surface adsorption. The activity-modulated HPO nanozyme induces a remarkable color tonality change from blue to yellow via synergistic modulation of dual absorbance values at 430 nm (increase) and 620 nm (decrease), achieving clear visual distinction. Using kanamycin as a model target, the ratiometric signal (ΔA₆₂₀/A₄₃₀) demonstrates significantly enhanced sensitivity (>6-fold improvement) compared to single-wavelength detection. The aptasensor further integrates smartphone-based RGB quantification to enable on-site analysis. Validation in milk and honey matrices confirms reliable recoveries (<7 % RSD), indicating their promising practical utility for real-samples detection. This HPO nanozyme platform advances multicolor ratiometric biosensing for on-site food safety monitoring, demonstrating broad applicability in antibiotic residue detection.